Bispecific Anti-BCMA x Anti-CD3 Antibodies and Uses Thereof

ABSTRACT

B-cell maturation antigen (BCMA) is expressed on malignant plasma cells. The present invention provides novel bispecific antibodies (bsAbs) that bind to both BCMA and CD3 and activate T cells via the CD3 complex in the presence of BCMA-expressing tumor cells. In certain embodiments, the bispecific antigen-binding molecules of the present invention are capable of inhibiting the growth of tumors expressing BCMA. The bispecific antigen-binding molecules of the invention are useful for the treatment of diseases and disorders in which an upregulated or induced BCMA-targeted immune response is desired and/or therapeutically beneficial. For example, the bispecific antibodies of the invention are useful for the treatment of various cancers, including multiple myeloma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e) of USProvisional Application Nos.: 62/700,596, filed Jul. 19, 2018;62/750,968, filed Oct. 26, 2018; and 62/793,645, filed Jan. 17, 2019,each of which is incorporated herein by reference in its entirety forall purposes.

REFERENCE TO A SEQUENCE LISTING

This application incorporates by reference the Sequence Listingsubmitted in Computer Readable Form as file 10452US01-Sequence, createdon Jul. 17, 2019 and containing 63,211 bytes.

FIELD OF THE INVENTION

The present invention relates to antibodies, and antigen-bindingfragments thereof, which are specific for B-cell maturation antigen(BCMA), and methods of use thereof. The present invention includesbispecific antigen-binding molecules (e.g., bispecific antibodies) thatbind BCMA and CD3, and methods of use thereof.

BACKGROUND

B-cell maturation antigen (BCMA), also known as TNFRSF17, or CD269, is atype III transmembrane protein lacking a signal peptide and containing acysteine-rich extracellular domain. BCMA, along with closely relatedproteins, promotes B-cell survival at distinct stages of development.BCMA is expressed exclusively in B-cell lineage cells, particularly inthe interfollicular region of the germinal center as well as onplasmablasts and differentiated plasma cells. BCMA is selectivelyinduced during plasma cell differentiation, and is required for optimalsurvival of long-lived plasma cells in the bone marrow. In multiplemyeloma, BCMA is widely expressed on malignant plasma cells at elevatedlevels, and BCMA expression is increased with progression from normalcells to active multiple myeloma. BCMA is also expressed in other B-cellmalignancies, including Waldenström's macroglobulinemia, Burkittlymphoma, and Diffuse Large B-Cell Lymphoma. Tai et al., Immunotherapy,7(11):1187-1199, 2015.

CD3 is a homodimeric or heterodimeric antigen expressed on T cells inassociation with the T cell receptor complex (TCR) and is required for Tcell activation. Functional CD3 is formed from the dimeric associationof two of four different chains: epsilon, zeta, delta and gamma. The CD3dimeric arrangements include gamma/epsilon, delta/epsilon and zeta/zeta.Antibodies against CD3 have been shown to duster CD3 on T cells, therebycausing T cell activation in a manner similar to the engagement of theTCR by peptide-loaded MHC molecules. Thus, anti-CD3 antibodies have beenproposed for therapeutic purposes involving the activation of T cells.In addition, bispecific antibodies that are capable of binding CD3 and atarget antigen have been proposed for therapeutic uses involvingtargeting T cell immune responses to tissues and cells expressing thetarget antigen.

Antigen-binding molecules that target BCMA, including bispecificantigen-binding molecules that bind both BCMA and CD3 would be useful intherapeutic settings in which specific targeting and T cell-mediatedkilling of cells that express BCMA is desired.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides an isolated bispecificantigen binding molecule comprising: (a) a first antigen-binding domainthat specifically binds a human B cell maturation antigen (BCMA) on atarget tumor cell, with an EC₅₀ of less than about 100 nM as measured byan in vitro FACS binding assay; and (b) a second antigen-binding domain(D2) that specifically binds human CD3 with an EC₅₀ of less than about10⁻⁶ M as measured by an in vitro FACS binding assay.

In some cases, the bispecific antigen binding molecule activates T cellsin vitro with an EC₅₀ of less than about 10⁻⁹ M. In some cases, thebispecific antigen-binding molecule mediates in vitro T cell killing oftumor cell lines expressing BCMA with an EC₅₀ of less than about 10⁻⁹ M.In some cases, the bispecific antigen-binding molecule mediates in vitroautologous T cell killing of primary myeloma cells expressing BCMA withan EC₅₀ of less than about 10 M. In some embodiments, the bispecificantigen-binding molecule interacts with amino acid residues 1 through 43of BCMA as set forth in SEQ ID NO: 115.

In some cases, the target tumor cell is a plasma cell. In some cases,the target tumor cell is from a patient suffering from multiple myeloma,or from another B-cell disorder characterized in part as having B cellsexpressing BCMA. In some cases, the bispecific antigen-binding moleculeinhibits the proliferation of BCMA expressing tumor cells at a dose offrom about 0.04 mg/kg to about 4.0 mg/kg. In some cases, the dose is0.04 mg/kg, 0.4 mg/kg or 4 mg/kg. In some embodiments, the dose isadministered to a patient in need thereof at least twice weekly for atleast seven doses. In some cases, the bispecific antigen-bindingmolecule inhibits the proliferation of BCMA+ tumor cells selected fromthe group consisting of myeloma cells, lymphoma cells and leukemiacells. In some cases, the bispecific antigen-binding molecule inhibitsthe proliferation of BCMA+ tumor cells selected from the groupconsisting of H929 cells, MOLP-8 cells and OPM cells.

In some cases, the bispecific antigen-binding molecule cross-reacts withcynomolgus BCMA. In some cases, the bispecific antigen-binding moleculedoes not cross-react with cynomolgus BCMA.

In some embodiments, the isolated bispecific antigen binding moleculecomprises a first antigen-binding domain that comprises: (a) three heavychain complementarity determining regions (HCDR1, HCDR2 and HCDR3)contained within a heavy chain variable region (HCVR) comprising theamino acid sequence of SEQ ID NO: 66; and (b) three light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3) containedwithin a light chain variable region (LCVR) comprising the amino acidsequence of SEQ ID NO:82. In some cases, the isolated bispecific antigenbinding molecule comprises a HCDR1 comprising the amino acid sequence ofSEQ ID NO:68, a HCDR2 comprising the amino acid sequence of SEQ IDNO:70, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:72.In some cases, the isolated bispecific antigen-binding moleculecomprises a LCDR1 comprising the amino acid sequence of SEQ ID NO:84, aLCDR2 comprising the amino acid sequence of SEQ ID NO:86, and a LCDR3comprising the amino acid sequence of SEQ ID NO:88. In some cases, thefirst antigen-binding domain comprises a HCVR comprising the amino acidsequence of SEQ ID NO: 66, and a LCVR comprising the amino acid sequenceof SEQ ID NO: 82.

In some embodiments, the isolated bispecific antigen-binding moleculecomprises a second antigen-binding domain that comprises: (a) threeheavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3)contained within a heavy chain variable region (HCVR) comprising theamino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 98; and (b) threelight chain complementarity determining regions (LCDR1, LCDR2 and LCDR3)contained within a light chain variable region (LCVR) comprising theamino acid sequence of SEQ ID NO:82. In some cases, the secondantigen-binding domain comprises: (a) a HCDR1 comprising the amino acidsequence of SEQ ID NO: 92 or SEQ ID NO: 100; (b) a HCDR2 comprising theamino acid sequence of SEQ ID NO: 94 or SEQ ID NO: 102; and (c) a HCDR3comprising the amino acid sequence of SEQ ID NO: 96 or SEQ ID NO: 104.In some cases, the second antigen-binding domain comprises a LCDR1comprising the amino acid sequence of SEQ ID NO:84, a LCDR2 comprisingthe amino acid sequence of SEQ ID NO:86, and a LCDR3 comprising theamino acid sequence of SEQ ID NO:88. In some cases, the secondantigen-binding domain comprises: (a) HCDR1, HCDR2, HCDR3 domains,respectively, comprising the amino acid sequences of SEQ ID NOs: 92, 94,96; and LCDR1, LCDR2, LCDR3 domains, respectively, comprising the aminoacid sequences of SEQ ID NOs: 84, 86, 88; or (b) HCDR1, HCDR2, HCDR3domains, respectively, comprising the amino acid sequences of SEQ IDNOs: 100, 102, 104; and LCDR1, LCDR2, LCDR3 domains, respectively,comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88. In somecases, the second antigen-binding domain comprises: (a) a HCVRcomprising the amino acid sequence of SEQ ID NO: 90, and a LCVRcomprising the amino acid sequence of SEQ ID NO: 82; or (b) a HCVRcomprising the amino acid sequence of SEQ ID NO: 98, and a LCVRcomprising the amino acid sequence of SEQ ID NO: 82.

In another aspect, the present invention provides an isolated bispecificantigen-binding molecule, comprising: (a) a first antigen-binding domainthat comprises HCDR1, HCDR2, HCDR3 domains, respectively, comprising theamino acid sequences of SEQ ID NOs: 68, 70, 72, and LCDR1, LCDR2, LCDR3domains, respectively, comprising the amino acid sequences of SEQ IDNOs: 84, 86, 88; and (b) a second antigen binding domain that comprisesHCDR1, HCDR2, HCDR3 domains, respectively, comprising the amino acidsequences of SEQ ID NOs: 92, 94, 96, and LCDR1, LCDR2, LCDR3 domains,respectively, comprising the amino acid sequences of SEQ ID NOs: 84, 86,88. In some cases, the isolated bispecific antigen-binding moleculecomprises: (a) a first antigen binding domain that comprises a HCVRcomprising the amino acid sequence of SEQ ID NO: 66, and a LCVRcomprising the amino acid sequence of SEQ ID NO: 82; and (b) a secondantigen binding domain that comprises a HCVR comprising the amino acidsequence of SEQ ID NO: 90, and a LCVR comprising the amino acid sequenceof SEQ ID NO: 82.

In another aspect, the present invention provides an isolated bispecificantigen-binding molecule, comprising: (a) a first antigen-binding domainthat comprises HCDR1, HCDR2, HCDR3 domains, respectively, comprising theamino acid sequences of SEQ ID NOs: 68, 70, 72, and LCDR1, LCDR2, LCDR3domains, respectively, comprising the amino acid sequences of SEQ IDNOs: 84, 86, 88; and (b) a second antigen binding domain that comprisesHCDR1, HCDR2, HCDR3 domains, respectively, comprising the amino acidsequences of SEQ ID NOs: 100, 102, 104, and LCDR1, LCDR2, LCDR3 domains,respectively, comprising the amino acid sequences of SEQ ID NOs: 84, 86,88. In some cases, the isolated bispecific antigen-binding moleculecomprises: (a) a first antigen binding domain that comprises a HCVRcomprising the amino acid sequence of SEQ ID NO: 66, and a LCVRcomprising the amino acid sequence of SEQ ID NO: 82; and (b) a secondantigen binding domain that comprises a HCVR comprising the amino acidsequence of SEQ ID NO: 98, and a LCVR comprising the amino acid sequenceof SEQ ID NO: 82.

In another aspect, the present invention provides an isolated bispecificantigen-binding molecule, comprising: (a) a first antigen-binding domainthat specifically binds human BCMA, and comprises the CDRs of a HCVRcomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2, 18, 34, 50, 66, 122, and 124, and the CDRs of a LCVRcomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 10, 26, 42, 58, 74, 82, 123, and 125; and (b) a secondantigen-binding domain that specifically binds human CD3. In some cases,the first antigen-binding domain comprises the CDRs from a HCVR/LCVRamino acid sequence pair selected from the group consisting of SEQ IDNOs: 2/10, 18/26, 34/42, 50/58, 66/74, 122/123, 124/125, 2/82, 18/82,34/82, 50/82, 66/82, 122/82, and 124/82. In some cases, the firstantigen-binding domain comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3domains, respectively, selected from the group consisting of SEQ ID NOs:4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64,68-70-72-76-78-80, 4-6-8-84-86-88, 20-22-24-84-86-88, 36-38-40-84-86-88,52-54-56-84-86-88, and 68-70-72-84-86-88. In some cases, the firstantigen-binding domain comprises the a HCVR/LCVR amino acid sequencepair selected from the group consisting of SEQ ID NOs: 2/10, 18/26,34/42, 50/58, 66/74, 122/123, 124/125, 2/82, 18/82, 34/82, 50/82, 66/82,122/82, and 124/82. In some cases, the second antigen-binding domaincomprises the CDRs of a HCVR/LCVR amino acid sequence pair selected fromthe group consisting of SEQ ID NOs: 90/82 and 98/82.

In another aspect, the present invention provides an isolated bispecificantigen binding molecule that competes for binding to BCMA, or binds tothe same epitope on BCMA as a reference antibody, wherein the referenceantibody comprises a first antigen-binding domain comprising anHCVR/LCVR pair comprising the amino acid sequences of SEQ ID NOs: 66/82and a second antigen-binding domain comprising an HCVR/LCVR paircomprising the amino acid sequences of either SEQ ID NOs: 90/82 or SEQID NOs: 98/82.

In another aspect, the present invention provides an isolated bispecificantigen binding molecule that competes for binding to human CD3, orbinds to the same epitope on human CD3 as a reference antibody, whereinthe reference antibody comprises a first antigen-binding domaincomprising an HCVR/LCVR pair comprising the amino acid sequences of SEQID NOs: 66/82 and a second antigen-binding domain comprising anHCVR/LCVR pair comprising the amino acid sequences of either SEQ ID NOs:90/82 or SEQ ID NOs: 98/82.

Any of the bispecific antigen-binding molecules discussed above orherein may be a bispecific antibody. In some cases, the bispecificantibody comprises a human IgG heavy chain constant region. In somecases, the human IgG heavy chain constant region is isotype IgG1. Insome cases, the human IgG heavy chain constant region is isotype IgG4.In various embodiments, the bispecific antibody comprises a chimerichinge that reduces Fcγ receptor binding relative to a wild-type hinge ofthe same isotype.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising the bispecific antigen-binding molecule (e.g.,bispecific antibody) discussed above or herein, and a pharmaceuticallyacceptable carrier or diluent.

In another aspect, the present invention provides a nucleic acidmolecule comprising a nucleotide sequence encoding a bispecificantigen-binding molecule (e.g., bispecific antibody) discussed above orherein.

In another aspect, the present invention provides an expression vectorcomprising the nucleic acid molecule discussed above.

In another aspect, the present invention provides a host cell comprisingthe expression vector discussed above.

In another aspect, the present invention provides a method of inhibitinggrowth of a plasma cell tumor in a subject, comprising administering anisolated bispecific antigen-binding molecule, or a pharmaceuticalcomposition comprising the bispecific antigen-binding molecule, asdiscussed above or herein, to the subject. In some cases, the plasmacell tumor is multiple myeloma. In some cases, the method furthercomprises administering a second therapeutic agent, or therapeuticregimen. In some embodiments, the second therapeutic agent comprises ananti-tumor agent (e.g. chemotherapeutic agents including melphalan,vincristine (Oncovin), cyclophosphamide (Cytoxan), etoposide (VP-16),doxorubicin (Adriamycin), liposomal doxorubicin (Doxil), obendamustine(Treanda), or any others known to be effective in treating a plasma celltumor in a subject). In some embodiments, the second therapeutic agentcomprises steroids. In some embodiments, the second therapeutic agentcomprises targeted therapies including thalidomide, lenalidomide, andbortezomib, which are therapies approved to treat newly diagnosedpatients. Lenalidomide, pomalidomide, bortezomib, carfilzomib,panobinostat, ixazomib, elotuzumab, and daratumumab are examples of asecond therapeutic agent effective for treating recurrent myeloma. Incertain embodiments the second therapeutic agent is a regimen comprisingradiotherapy or a stem cell transplant. In certain embodiments, thesecond therapeutic agent may be an immunomodulatory agent. In certainembodiments, the second therapeutic agent may be a proteasome inhibitor,including bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib(Ninlaro). In certain embodiments the second therapeutic agent may be ahistone deacetylase inhibitor such as panobinostat (Farydak). In certainembodiments, the second therapeutic agent may be a monoclonal antibody,an antibody drug conjugate, a bispecific antibody conjugated to ananti-tumor agent, a checkpoint inhibitor, or combinations thereof.

In another aspect, the present invention provides a method of treating apatient suffering from multiple myeloma, or from another BCMA-expressingB cell malignancy, where the method comprises administering an isolatedbispecific antigen-binding molecule or a pharmaceutical compositioncomprising the bispecific antigen-binding molecule, as discussed aboveor herein, to the subject. In some cases, the BCMA-expressing B cellmalignancy is selected from the group consisting of Waldenström'smacroglobulinemia, Burkitt's lymphoma and Diffuse Large B-Cell lymphoma,Non-Hodgkin's lymphoma, chronic lymphocytic leukemia, follicularlymphoma, mantle cell lymphoma, marginal zone lymphoma,lymphoplasmacytic lymphoma, and Hodgkin's lymphoma. In some cases, themethod further comprises administering a second therapeutic agent. Insome embodiments, the second therapeutic agent comprises an anti-tumoragent (a chemotherapeutic agent), DNA alkylators, immunomodulators,proteasome inhibitors, histone deacetylase inhibitors radiotherapy, astem cell transplant, an immunomodulator, a monoclonal antibody thatinteracts with an antigen expressed on the tumor cell surface, amonoclonal antibody other than those described herein, which mayinteract with a different antigen on the plasma cell surface, abispecific antibody, which has one arm that binds to an antigen on thetumor cell surface and the other arm binds to an antigen on a T cell, anantibody drug conjugate, a bispecific antibody conjugated with ananti-tumor agent, a checkpoint inhibitor, for example, one that targets,PD-1 or CTLA-4, or combinations thereof. In certain embodiments, thecheckpoint inhibitors may be selected from PD-1 inhibitors, such aspembrolizumab (Keytruda), nivolumab (Opdivo), or cemiplimab (REGN2810).In certain embodiments, the checkpoint inhibitors may be selected fromPD-L1 inhibitors, such as atezolizumab (Tecentriq), avelumab (Bavencio),or Durvalumab (Imfinzi)). In certain embodiments, the checkpointinhibitors may be selected from CTLA-4 inhibitors, such as ipilimumab(Yervoy). Other combinations that may be used in conjunction with anantibody of the invention are described above.

In another aspect, the present invention provides a method of treating apatient suffering from a BCMA-expressing tumor, wherein the methodcomprises administering an isolated bispecific antigen-binding moleculeas discussed above or herein, or a pharmaceutical composition comprisingsame, to the subject in combination with an anti-PD-1 antibody orantigen-binding fragment thereof. In some cases, the anti-PD-1 antibodyor antigen-binding fragment is an anti-PD-1 antibody. In someembodiments, the anti-PD-1 antibody is cemiplimab (REGN2810). In variousembodiments, the combination of anti-BCMA x anti-CD3 bispecificantigen-binding molecule (e.g., a bispecific antibody) and an anti-PD-1antibody or antigen-binding fragment (e.g., an anti-PD-1 antibody)produces a synergistic therapeutic effect in the treatment ofBCMA-expressing tumors.

In another aspect, the present invention provides for use of thebispecific antigen-binding molecules discussed above or herein, or thepharmaceutical compositions discussed above or herein, in the treatmentof a disease or disorder associated with expression of BCMA. In somecases, the disease or disorder is cancer. In some embodiments, thecancer is multiple myeloma. In some cases, the disease or disorder isCastleman disease. In some cases, the antigen-binding molecules are foruse in combination with an anti-PD-1 antibody or antigen-bindingfragment thereof, optionally wherein the anti-PD-1 antibody iscemiplimab (REGN2810).

The present invention further includes use of the bispecificantigen-binding molecules discussed above or herein in the manufactureof a medicament for treating a disease or disorder associated withexpression of BCMA. In some cases, the disease or disorder is cancer. Insome embodiments, the cancer is multiple myeloma.

In various embodiments, any of the features or components of embodimentsdiscussed above or herein may be combined, and such combinations areencompassed within the scope of the present disclosure. Any specificvalue discussed above or herein may be combined with another relatedvalue discussed above or herein to recite a range with the valuesrepresenting the upper and lower ends of the range, and such ranges areencompassed within the scope of the present disclosure.

Other embodiments will become apparent from a review of the ensuingdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 illustrate prophylactic dose-dependent tumor inhibition ofBCMA-expressing NCI-H929 human multiple myeloma tumor cells in vivo byanti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459,respectively. NCI-H929 cells express high levels of BCMA.

FIGS. 3 and 4 illustrate therapeutic dose-dependent tumor inhibition ofestablished BCMA-expressing NCI-H929 human multiple myeloma tumor cellsin vivo by anti-BCMA x anti-CD3 bispecific antibodies REGN5458 andREGN5459, respectively. NCI-H929 cells express high levels of BCMA.

FIGS. 5 and 6 illustrate prophylactic dose-dependent tumor inhibition ofBCMA-expressing MOLP-8 human multiple myeloma tumor cells in vivo byanti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459,respectively. MOLP-8 cells express moderate levels of BCMA.

FIG. 7 illustrates a therapeutic reduction in established tumor burdenof BCMA-expressing OPM-2 human multiple myeloma tumor cells in vivo byanti-BCMA x anti-CD3 bispecific antibodies REGN5458 and REGN5459,relative to controls. OPM-2 cells express low levels of BCMA.

DETAILED DESCRIPTION

Before the present invention is described, it is to be understood thatthis invention is not limited to particular methods and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein, the term“about,” when used in reference to a particular recited numerical value,means that the value may vary from the recited value by no more than 1%.For example, as used herein, the expression “about 100” includes 99 and101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described. Allpatents, applications and non-patent publications mentioned in thisspecification are incorporated herein by reference in their entireties.

Definitions

The expression “CD3,” as used herein, refers to an antigen which isexpressed on T cells as part of the multimolecular T cell receptor (TCR)and which consists of a homodimer or heterodimer formed from theassociation of two of four receptor chains: CD3-epsilon, CD3-delta,CD3-zeta, and CD3-gamma. Human CD3-epsilon comprises the amino acidsequence as set forth in SEQ ID NO:116; human CD3-delta comprises theamino acid sequence as set forth in SEQ ID NO:117; human CD3-zetacomprises the amino acid sequence as set forth in SEQ ID NO: 118; andCD3-gamma comprises the amino acid sequence as set forth in SEQ ID NO119. All references to proteins, polypeptides and protein fragmentsherein are intended to refer to the human version of the respectiveprotein, polypeptide or protein fragment unless explicitly specified asbeing from a non-human species. Thus, the expression “CD3” means humanCD3 unless specified as being from a non-human species, e.g., “mouseCD3,” “monkey CD3,” etc.

As used herein, “an antibody that binds CD3” or an “anti-CD3 antibody”includes antibodies and antigen-binding fragments thereof thatspecifically recognize a single CD3 subunit (e.g., epsilon, delta, gammaor zeta), as well as antibodies and antigen-binding fragments thereofthat specifically recognize a dimeric complex of two CD3 subunits (e.g.,gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers). The antibodiesand antigen-binding fragments of the present invention may bind solubleCD3 and/or cell surface expressed CD3. Soluble CD3 includes natural CD3proteins as well as recombinant CD3 protein variants such as, e.g.,monomeric and dimeric CD3 constructs, that lack a transmembrane domainor are otherwise unassociated with a cell membrane.

As used herein, the expression “cell surface-expressed CD3” means one ormore CD3 protein(s) that is/are expressed on the surface of a cell invitro or in vivo, such that at least a portion of a CD3 protein isexposed to the extracellular side of the cell membrane and is accessibleto an antigen-binding portion of an antibody. “Cell surface-expressedCD3” includes CD3 proteins contained within the context of a functionalT cell receptor in the membrane of a cell. The expression “cellsurface-expressed CD3” includes CD3 protein expressed as part of ahomodimer or heterodimer on the surface of a cell (e.g., gamma/epsilon,delta/epsilon, and zeta/zeta CD3 dimers). The expression, “cellsurface-expressed CD3” also includes a CD3 chain (e.g., CD3-epsilon,CD3-delta or CD3-gamma) that is expressed by itself, without other CD3chain types, on the surface of a cell. A “cell surface-expressed CD3”can comprise or consist of a CD3 protein expressed on the surface of acell which normally expresses CD3 protein. Alternatively, “cellsurface-expressed CD3” can comprise or consist of CD3 protein expressedon the surface of a cell that normally does not express human CD3 on itssurface but has been artificially engineered to express CD3 on itssurface.

The expression “BCMA,” as used herein, refers to B-cell maturationantigen. BCMA (also known as TNFRSF17 and CD269) is a cell surfaceprotein expressed on malignant plasma cells, and plays a central role inregulating B cell maturation and differentiation intoimmunoglobulin-producing plasma cells. The amino acid sequence of humanBCMA is shown in SEQ ID NO: 115, and can also be found in GenBankaccession number NP_001183.2.

As used herein, “an antibody that binds BCMA” or an “anti-BCMA antibody”includes antibodies and antigen-binding fragments thereof thatspecifically recognize BCMA.

The term “antigen-binding molecule” includes antibodies andantigen-binding fragments of antibodies, including, e.g., bispecificantibodies.

The term “antibody”, as used herein, means any antigen-binding moleculeor molecular complex comprising at least one complementarity determiningregion (CDR) that specifically binds to or interacts with a particularantigen (e.g., BCMA or CD3). The term “antibody” includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM). The term “antibody” also includesimmunoglobulin molecules consisting of four polypeptide chains, twoheavy (H) chains and two light (L) chains inter-connected by disulfidebonds. Each heavy chain comprises a heavy chain variable region(abbreviated herein as HCVR or V_(H)) and a heavy chain constant region.The heavy chain constant region comprises three domains, C_(H)1, C_(H)2and C_(H)3. Each light chain comprises a light chain variable region(abbreviated herein as LCVR or V_(L)) and a light chain constant region.The light chain constant region comprises one domain (C_(L)1). The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments ofthe invention, the FRs of the anti-BCMA antibody or anti-CD3 antibody(or antigen-binding portion thereof) may be identical to the humangermline sequences, or may be naturally or artificially modified. Anamino acid consensus sequence may be defined based on a side-by-sideanalysis of two or more CDRs.

The term “antibody”, as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

An antigen-binding fragment of an antibody will typically comprise atleast one variable domain. The variable domain may be of any size oramino acid composition and will generally comprise at least one CDRwhich is adjacent to or in frame with one or more framework sequences.In antigen-binding fragments having a V_(H) domain associated with aV_(L) domain, the V_(H) and V_(L) domains may be situated relative toone another in any suitable arrangement. For example, the variableregion may be dimeric and contain V_(H)—V_(H), V_(H)—V_(L) orV_(L)—V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (v)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-C_(L);(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2; (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments may bemonospecific or multispecific (e.g., bispecific). A multispecificantigen-binding fragment of an antibody will typically comprise at leasttwo different variable domains, wherein each variable domain is capableof specifically binding to a separate antigen or to a different epitopeon the same antigen. Any multispecific antibody format, including theexemplary bispecific antibody formats disclosed herein, may be adaptedfor use in the context of an antigen-binding fragment of an antibody ofthe present invention using routine techniques available in the art.

The antibodies of the present invention may function throughcomplement-dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC). “Complement-dependent cytotoxicity”(CDC) refers to lysis of antigen-expressing cells by an antibody of theinvention in the presence of complement. “Antibody-dependentcell-mediated cytotoxicity” (ADCC) refers to a cell-mediated reaction inwhich nonspecific cytotoxic cells that express Fc receptors (FcRs)(e.g., Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and thereby lead to lysis ofthe target cell. CDC and ADCC can be measured using assays that are wellknown and available in the art. (See, e.g., U.S. Pat. Nos. 5,500,362 and5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA)95:652-656). The constant region of an antibody is important in theability of an antibody to fix complement and mediate cell-dependentcytotoxicity. Thus, the isotype of an antibody may be selected on thebasis of whether it is desirable for the antibody to mediatecytotoxicity.

In certain embodiments of the invention, the anti-BCMA monospecificantibodies or anti-BCMA x anti-CD3 bispecific antibodies of theinvention are human antibodies. The term “human antibody”, as usedherein, is intended to include antibodies having variable and constantregions derived from human germline immunoglobulin sequences. The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo), for example in the CDRs and in particular CDR3. However, theterm “human antibody”, as used herein, is not intended to includeantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

The antibodies of the invention may, in some embodiments, be recombinanthuman antibodies. The term “recombinant human antibody”, as used herein,is intended to include all human antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodiesexpressed using a recombinant expression vector transfected into a hostcell (described further below), antibodies isolated from a recombinant,combinatorial human antibody library (described further below),antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl.Acids Res. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivesomatic mutagenesis) and thus the amino acid sequences of the V_(H) andV_(L) regions of the recombinant antibodies are sequences that, whilederived from and related to human germline V_(H) and V_(L) sequences,may not naturally exist within the human antibody germline repertoire invivo.

Human antibodies can exist in two forms that are associated with hingeheterogeneity. In one form, an immunoglobulin molecule comprises astable four chain construct of approximately 150-160 kDa in which thedimers are held together by an interchain heavy chain disulfide bond. Ina second form, the dimers are not linked via inter-chain disulfide bondsand a molecule of about 75-80 kDa is formed composed of a covalentlycoupled light and heavy chain (half-antibody). These forms have beenextremely difficult to separate, even after affinity purification.

The frequency of appearance of the second form in various intact IgGisotypes is due to, but not limited to, structural differencesassociated with the hinge region isotype of the antibody. A single aminoacid substitution in the hinge region of the human IgG4 hinge cansignificantly reduce the appearance of the second form (Angal et al.(1993) Molecular Immunology 30:105) to levels typically observed using ahuman IgG1 hinge. The instant invention encompasses antibodies havingone or more mutations in the hinge, C_(H)2 or C_(H)3 region which may bedesirable, for example, in production, to improve the yield of thedesired antibody form.

The antibodies of the invention may be isolated antibodies. An “isolatedantibody,” as used herein, means an antibody that has been identifiedand separated and/or recovered from at least one component of itsnatural environment. For example, an antibody that has been separated orremoved from at least one component of an organism, or from a tissue orcell in which the antibody naturally exists or is naturally produced, isan “isolated antibody” for purposes of the present invention. Anisolated antibody also includes an antibody in situ within a recombinantcell. Isolated antibodies are antibodies that have been subjected to atleast one purification or isolation step. According to certainembodiments, an isolated antibody may be substantially free of othercellular material and/or chemicals.

The present invention also includes one-arm antibodies that bind BCMA.As used herein, a “one-arm antibody” means an antigen-binding moleculecomprising a single antibody heavy chain and a single antibody lightchain. The one-arm antibodies of the present invention may comprise anyof the HCVR/LCVR or CDR amino acid sequences as set forth in Table 1.

The anti-BCMA or anti-BCMA x anti-CD3 antibodies disclosed herein maycomprise one or more amino acid substitutions, insertions and/ordeletions in the framework and/or CDR regions of the heavy and lightchain variable domains as compared to the corresponding germlinesequences from which the antibodies were derived. Such mutations can bereadily ascertained by comparing the amino acid sequences disclosedherein to germline sequences available from, for example, publicantibody sequence databases. The present invention includes antibodies,and antigen-binding fragments thereof, which are derived from any of theamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antibody was derived. In otherembodiments, only certain residues are mutated back to the originalgermline sequence, e.g., only the mutated residues found within thefirst 8 amino acids of FR1 or within the last 8 amino acids of FR4, oronly the mutated residues found within CDR1, CDR2 or CDR3. In otherembodiments, one or more of the framework and/or CDR residue(s) aremutated to the corresponding residue(s) of a different germline sequence(i.e., a germline sequence that is different from the germline sequencefrom which the antibody was originally derived). Furthermore, theantibodies of the present invention may contain any combination of twoor more germline mutations within the framework and/or CDR regions,e.g., wherein certain individual residues are mutated to thecorresponding residue of a particular germline sequence while certainother residues that differ from the original germline sequence aremaintained or are mutated to the corresponding residue of a differentgermline sequence. Once obtained, antibodies and antigen-bindingfragments that contain one or more germline mutations can be easilytested for one or more desired property such as, improved bindingspecificity, increased binding affinity, improved or enhancedantagonistic or agonistic biological properties (as the case may be),reduced immunogenicity, etc. Antibodies and antigen-binding fragmentsobtained in this general manner are encompassed within the presentinvention.

The present invention also includes anti-BCMA or anti-BCMA x anti-CD3antibodies comprising variants of any of the HCVR, LCVR, and/or CDRamino acid sequences disclosed herein having one or more conservativesubstitutions. For example, the present invention includes anti-BCMA oranti-BCMA x anti-CD3 antibodies having HCVR, LCVR, and/or CDR amino acidsequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer,etc. conservative amino acid substitutions relative to any of the HCVR,LCVR, and/or CDR amino acid sequences set forth in Tables 1 and 3herein, or the anti-CD3 antibodies disclosed in WO 2014/047231 or WO2017/053856, each of which is incorporated herein by reference.

The term “epitope” refers to an antigenic determinant that interactswith a specific antigen binding site in the variable region of anantibody molecule known as a paratope. A single antigen may have morethan one epitope. Thus, different antibodies may bind to different areason an antigen and may have different biological effects. Epitopes may beeither conformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstance, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

The term “substantial identity” or “substantially identical,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95%, and more preferablyat least about 96%, 97%, 98% or 99% of the nucleotide bases, as measuredby any well-known algorithm of sequence identity, such as FASTA, BLASTor Gap, as discussed below. A nucleic acid molecule having substantialidentity to a reference nucleic acid molecule may, in certain instances,encode a polypeptide having the same or substantially similar amino acidsequence as the polypeptide encoded by the reference nucleic acidmolecule.

As applied to polypeptides, the term “substantial similarity” or“substantially similar” means that two peptide sequences, when optimallyaligned, such as by the programs GAP or BESTFIT using default gapweights, share at least 95% sequence identity, even more preferably atleast 98% or 99% sequence identity. Preferably, residue positions whichare not identical differ by conservative amino acid substitutions. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chain(R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See, e.g., Pearson (1994)Methods Mol. Biol. 24: 307-331, herein incorporated by reference.Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartate and glutamate, and (7) sulfur-containingside chains are cysteine and methionine. Preferred conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine,glutamate-aspartate, and asparagine-glutamine. Alternatively, aconservative replacement is any change having a positive value in thePAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science256: 1443-1445, herein incorporated by reference. A “moderatelyconservative” replacement is any change having a nonnegative value inthe PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402, each herein incorporated byreference.

Germline Mutations

The anti-CD3 antibodies disclosed herein comprise one or more amino acidsubstitutions, insertions and/or deletions in the framework and/or CDRregions of the heavy chain variable domains as compared to thecorresponding germline sequences from which the antibodies were derived.

The present invention also includes antibodies, and antigen-bindingfragments thereof, which are derived from any of the amino acidsequences disclosed herein, wherein one or more amino acids within oneor more framework and/or CDR regions are mutated to the correspondingresidue(s) of the germline sequence from which the antibody was derived,or to the corresponding residue(s) of another human germline sequence,or to a conservative amino acid substitution of the correspondinggermline residue(s) (such sequence changes are referred to hereincollectively as “germline mutations”), and having weak or no detectablebinding to a CD3 antigen.

Furthermore, the antibodies of the present invention may contain anycombination of two or more germline mutations within the frameworkand/or CDR regions, e.g., wherein certain individual residues aremutated to the corresponding residue of a particular germline sequencewhile certain other residues that differ from the original germlinesequence are maintained or are mutated to the corresponding residue of adifferent germline sequence. Once obtained, antibodies andantigen-binding fragments that contain one or more germline mutationscan be tested for one or more desired properties such as, improvedbinding specificity, weak or reduced binding affinity, improved orenhanced pharmacokinetic properties, reduced immunogenicity, etc.Antibodies and antigen-binding fragments obtained in this general mannergiven the guidance of the present disclosure are encompassed within thepresent invention.

The present invention also includes antigen-binding molecules comprisingan antigen-binding domain with an HCVR and/or CDR amino acid sequencethat is substantially identical to any of the HCVR and/or CDR amino acidsequences disclosed herein, while maintaining or improving the desiredweak affinity to CD3 antigen. The term “substantial identity” or“substantially identical,” when referring to an amino acid sequencemeans that two amino acid sequences, when optimally aligned, such as bythe programs GAP or BESTFIT using default gap weights, share at least95% sequence identity, even more preferably at least 98% or 99% sequenceidentity. Preferably, residue positions which are not identical differby conservative amino acid substitutions. In cases where two or moreamino acid sequences differ from each other by conservativesubstitutions, the percent sequence identity or degree of similarity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG software contains programs such as Gap and Bestfitwhich can be used with default parameters to determine sequence homologyor sequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (2000) supra). Another preferred algorithm when comparing asequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters. See, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al.(1997) Nucleic Acids Res. 25:3389-402.

Binding Properties of the Antibodies

As used herein, the term “binding” in the context of the binding of anantibody, immunoglobulin, antibody-binding fragment, or Fc-containingprotein to either, e.g., a predetermined antigen, such as a cell surfaceprotein or fragment thereof, typically refers to an interaction orassociation between a minimum of two entities or molecular structures,such as an antibody-antigen interaction.

For instance, binding affinity typically corresponds to a K_(D) value ofabout 10⁻⁷ M or less, such as about 10⁻⁸ M or less, such as about 10⁻⁹ Mor less when determined by, for instance, surface plasmon resonance(SPR) technology in a BIAcore 3000 instrument using the antigen as theligand and the antibody, Ig, antibody-binding fragment, or Fc-containingprotein as the analyte (or antiligand). Cell-based binding strategies,such as fluorescent-activated cell sorting (FACS) binding assays, arealso routinely used, and FACS data correlates well with other methodssuch as radioligand competition binding and SPR (Benedict, C A, JImmunol Methods. 1997, 201(2):223-31; Geuijen, C A, et al. J ImmunolMethods. 2005, 302(1-2):68-77).

Accordingly, the antibody or antigen-binding protein of the inventionbinds to the predetermined antigen or cell surface molecule (receptor)having an affinity corresponding to a K_(D) value that is at leastten-fold lower than its affinity for binding to a non-specific antigen(e.g., BSA, casein). According to the present invention, the affinity ofan antibody corresponding to a K_(D) value that is equal to or less thanten-fold lower than a non-specific antigen may be considerednon-detectable binding, however such an antibody may be paired with asecond antigen binding arm for the production of a bispecific antibodyof the invention.

The term “K_(D)” (M) refers to the dissociation equilibrium constant ofa particular antibody-antigen interaction, or the dissociationequilibrium constant of an antibody or antibody-binding fragment bindingto an antigen. There is an inverse relationship between K_(D) andbinding affinity, therefore the smaller the K_(D) value, the higher,i.e. stronger, the affinity. Thus, the terms “higher affinity” or“stronger affinity” relate to a higher ability to form an interactionand therefore a smaller K_(D) value, and conversely the terms “loweraffinity” or “weaker affinity” relate to a lower ability to form aninteraction and therefore a larger K_(D) value. In some circumstances, ahigher binding affinity (or K_(D)) of a particular molecule (e.g.antibody) to its interactive partner molecule (e.g. antigen X) comparedto the binding affinity of the molecule (e.g. antibody) to anotherinteractive partner molecule (e.g. antigen Y) may be expressed as abinding ratio determined by dividing the larger K_(D) value (lower, orweaker, affinity) by the smaller K_(D) (higher, or stronger, affinity),for example expressed as 5-fold or 10-fold greater binding affinity, asthe case may be.

The term “k_(d)” (sec−1 or 1/s) refers to the dissociation rate constantof a particular antibody-antigen interaction, or the dissociation rateconstant of an antibody or antibody-binding fragment. Said value is alsoreferred to as the k_(off) value.

The term “k_(a)” (M−1×sec−1 or 1/M) refers to the association rateconstant of a particular antibody-antigen interaction, or theassociation rate constant of an antibody or antibody-binding fragment.

The term “K_(A)” (M−1 or 1/M) refers to the association equilibriumconstant of a particular antibody-antigen interaction, or theassociation equilibrium constant of an antibody or antibody-bindingfragment. The association equilibrium constant is obtained by dividingthe k, by the k_(d).

The term “EC50” or “EC₅₀” refers to the half maximal effectiveconcentration, which includes the concentration of an antibody whichinduces a response halfway between the baseline and maximum after aspecified exposure time. The EC₅₀ essentially represents theconcentration of an antibody where 50% of its maximal effect isobserved. In certain embodiments, the EC₅₀ value equals theconcentration of an antibody of the invention that gives half-maximalbinding to cells expressing CD3 or tumor-associated antigen (e.g.,BCMA), as determined by e.g. a FACS binding assay. Thus, reduced orweaker binding is observed with an increased EC₅₀, or half maximaleffective concentration value.

In one embodiment, decreased binding can be defined as an increased EC₅₀antibody concentration which enables binding to the half-maximal amountof target cells.

In another embodiment, the EC₅₀ value represents the concentration of anantibody of the invention that elicits half-maximal depletion of targetcells by T cell cytotoxic activity. Thus, increased cytotoxic activity(e.g. T cell-mediated tumor cell killing) is observed with a decreasedEC₅₀, or half maximal effective concentration value.

Bispecific Antigen-Binding Molecules

The antibodies of the present invention may be monospecific,bi-specific, or multispecific. Multispecific antibodies may be specificfor different epitopes of one target polypeptide or may containantigen-binding domains specific for more than one target polypeptide.See, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244. The anti-BCMA monospecific antibodies oranti-BCMA x anti-CD3 bispecific antibodies of the present invention canbe linked to or co-expressed with another functional molecule, e.g.,another peptide or protein. For example, an antibody or fragment thereofcan be functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody or antibody fragment to produce abi-specific or a multispecific antibody with a second or additionalbinding specificity.

Use of the expression “anti-CD3 antibody” or “anti-BCMA antibody” hereinis intended to include both monospecific anti-CD3 or anti-BCMAantibodies as well as bispecific antibodies comprising a CD3-binding armand a BCMA-binding arm. Thus, the present invention includes bispecificantibodies wherein one arm of an immunoglobulin binds human CD3, and theother arm of the immunoglobulin is specific for human BCMA. TheCD3-binding arm can comprise any of the HCVR/LCVR or CDR amino acidsequences as set forth in Table 3 herein, or the anti-CD3 antibodiesdisclosed in WO 2014/047231 or WO 2017/053856.

In certain embodiments, the CD3-binding arm binds to human CD3 andinduces human T cell activation. In certain embodiments, the CD3-bindingarm binds weakly to human CD3 and induces human T cell activation. Inother embodiments, the CD3-binding arm binds weakly to human CD3 andinduces tumor-associated antigen-expressing cell killing in the contextof a bispecific or multispecific antibody. In other embodiments, theCD3-binding arm binds or associates weakly with human and cynomolgus(monkey) CD3, yet the binding interaction is not detectable by in vitroassays known in the art. The BCMA-binding arm can comprise any of theHCVR/LCVR or CDR amino acid sequences as set forth in Table 1 herein.

According to certain exemplary embodiments, the present inventionincludes bispecific antigen-binding molecules that specifically bind CD3and BCMA. Such molecules may be referred to herein as, e.g., “anti-BCMAx anti-CD3” or “anti-CD3/anti-BCMA,” or “anti-CD3xBCMA” or “CD3xBCMA”bispecific molecules, or other similar terminology (e.g.,anti-BCMA/anti-CD3).

The term “BCMA,” as used herein, refers to the human BCMA protein unlessspecified as being from a non-human species (e.g., “mouse BCMA,” “monkeyBCMA,” etc.). The human BCMA protein has the amino acid sequence shownin SEQ ID NO: 115.

The aforementioned bispecific antigen-binding molecules thatspecifically bind CD3 and BCMA may comprise an anti-CD3 antigen-bindingmolecule which binds to CD3 with a weak binding affinity such asexhibiting a K_(D) of greater than about 40 nM, as measured by an invitro affinity binding assay.

As used herein, the expression “antigen-binding molecule” means aprotein, polypeptide or molecular complex comprising or consisting of atleast one complementarity determining region (CDR) that alone, or incombination with one or more additional CDRs and/or framework regions(FRs), specifically binds to a particular antigen. In certainembodiments, an antigen-binding molecule is an antibody or a fragment ofan antibody, as those terms are defined elsewhere herein.

As used herein, the expression “bispecific antigen-binding molecule”means a protein, polypeptide or molecular complex comprising at least afirst antigen-binding domain and a second antigen-binding domain. Eachantigen-binding domain within the bispecific antigen-binding moleculecomprises at least one CDR that alone, or in combination with one ormore additional CDRs and/or FRs, specifically binds to a particularantigen. In the context of the present invention, the firstantigen-binding domain specifically binds a first antigen (e.g., BCMA),and the second antigen-binding domain specifically binds a second,distinct antigen (e.g., CD3).

In certain exemplary embodiments of the present invention, thebispecific antigen-binding molecule is a bispecific antibody. Eachantigen-binding domain of a bispecific antibody comprises a heavy chainvariable domain (HCVR) and a light chain variable domain (LCVR). In thecontext of a bispecific antigen-binding molecule comprising a first anda second antigen-binding domain (e.g., a bispecific antibody), the CDRsof the first antigen-binding domain may be designated with the prefix“D1” and the CDRs of the second antigen-binding domain may be designatedwith the prefix “D2”. Thus, the CDRs of the first antigen-binding domainmay be referred to herein as D1-HCDR1, D1-HCDR2, and D1-HCDR3; and theCDRs of the second antigen-binding domain may be referred to herein asD2-HCDR1, D2-HCDR2, and D2-HCDR3.

In certain exemplary embodiments, the isolated bispecific antigenbinding molecule comprises a first antigen-binding domain thatcomprises: (a) three heavy chain complementarity determining regions(HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region(HCVR) comprising the amino acid sequence of SEQ ID NO: 66; and (b)three light chain complementarity determining regions (LCDR1, LCDR2 andLCDR3) contained within a light chain variable region (LCVR) comprisingthe amino acid sequence of SEQ ID NO:82. In some cases, the isolatedbispecific antigen binding molecule comprises a HCDR1 comprising theamino acid sequence of SEQ ID NO:68, a HCDR2 comprising the amino acidsequence of SEQ ID NO:70, and a HCDR3 comprising the amino acid sequenceof SEQ ID NO:72. In some cases, the isolated bispecific antigen-bindingmolecule comprises a LCDR1 comprising the amino acid sequence of SEQ IDNO:84, a LCDR2 comprising the amino acid sequence of SEQ ID NO:86, and aLCDR3 comprising the amino acid sequence of SEQ ID NO:88. In some cases,the first antigen-binding domain comprises a HCVR comprising the aminoacid sequence of SEQ ID NO: 66, and a LCVR comprising the amino acidsequence of SEQ ID NO: 82.

In certain exemplary embodiments, the isolated bispecificantigen-binding molecule comprises a second antigen-binding domain thatcomprises: (a) three heavy chain complementarity determining regions(HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region(HCVR) comprising the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO:98; and (b) three light chain complementarity determining regions(LCDR1, LCDR2 and LCDR3) contained within a light chain variable region(LCVR) comprising the amino acid sequence of SEQ ID NO:82. In somecases, the second antigen-binding domain comprises: (a) a HCDR1comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 100;(b) a HCDR2 comprising the amino acid sequence of SEQ ID NO: 94 or SEQID NO: 102; and (c) a HCDR3 comprising the amino acid sequence of SEQ IDNO: 96 or SEQ ID NO: 104. In some cases, the second antigen-bindingdomain comprises a LCDR1 comprising the amino acid sequence of SEQ IDNO:84, a LCDR2 comprising the amino acid sequence of SEQ ID NO:86, and aLCDR3 comprising the amino acid sequence of SEQ ID NO:88. In some cases,the second antigen-binding domain comprises: (a) HCDR1, HCDR2, HCDR3domains, respectively, comprising the amino acid sequences of SEQ IDNOs: 92, 94, 96; and LCDR1, LCDR2, LCDR3 domains, respectively,comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88; or (b)HCDR1, HCDR2, HCDR3 domains, respectively, comprising the amino acidsequences of SEQ ID NOs: 100, 102, 104; and LCDR1, LCDR2, LCDR3 domains,respectively, comprising the amino acid sequences of SEQ ID NOs: 84, 86,88. In some cases, the second antigen-binding domain comprises: (a) aHCVR comprising the amino acid sequence of SEQ ID NO: 90, and a LCVRcomprising the amino acid sequence of SEQ ID NO: 82; or (b) a HCVRcomprising the amino acid sequence of SEQ ID NO: 98, and a LCVRcomprising the amino acid sequence of SEQ ID NO: 82.

In certain exemplary embodiments, the isolated bispecificantigen-binding molecule comprises: (a) a first antigen-binding domainthat comprises HCDR1, HCDR2, HCDR3 domains, respectively, comprising theamino acid sequences of SEQ ID NOs: 68, 70, 72, and LCDR1, LCDR2, LCDR3domains, respectively, comprising the amino acid sequences of SEQ IDNOs: 84, 86, 88; and (b) a second antigen binding domain that comprisesHCDR1, HCDR2, HCDR3 domains, respectively, comprising the amino acidsequences of SEQ ID NOs: 92, 94, 96, and LCDR1, LCDR2, LCDR3 domains,respectively, comprising the amino acid sequences of SEQ ID NOs: 84, 86,88. In some cases, the isolated bispecific antigen-binding moleculecomprises: (a) a first antigen binding domain that comprises a HCVRcomprising the amino acid sequence of SEQ ID NO: 66, and a LCVRcomprising the amino acid sequence of SEQ ID NO: 82; and (b) a secondantigen binding domain that comprises a HCVR comprising the amino acidsequence of SEQ ID NO: 90, and a LCVR comprising the amino acid sequenceof SEQ ID NO: 82.

In certain exemplary embodiments, the isolated bispecificantigen-binding molecule comprises: (a) a first antigen-binding domainthat comprises HCDR1, HCDR2, HCDR3 domains, respectively, comprising theamino acid sequences of SEQ ID NOs: 68, 70, 72, and LCDR1, LCDR2, LCDR3domains, respectively, comprising the amino acid sequences of SEQ IDNOs: 84, 86, 88; and (b) a second antigen binding domain that comprisesHCDR1, HCDR2, HCDR3 domains, respectively, comprising the amino acidsequences of SEQ ID NOs: 100, 102, 104, and LCDR1, LCDR2, LCDR3 domains,respectively, comprising the amino acid sequences of SEQ ID NOs: 84, 86,88. In some cases, the isolated bispecific antigen-binding moleculecomprises: (a) a first antigen binding domain that comprises a HCVRcomprising the amino acid sequence of SEQ ID NO: 66, and a LCVRcomprising the amino acid sequence of SEQ ID NO: 82; and (b) a secondantigen binding domain that comprises a HCVR comprising the amino acidsequence of SEQ ID NO: 98, and a LCVR comprising the amino acid sequenceof SEQ ID NO: 82.

In certain exemplary embodiments, the isolated bispecificantigen-binding molecule comprises: (a) a first antigen-binding domainthat specifically binds human BCMA, and comprises the CDRs of a HCVRcomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2, 18, 34, 50, 66, 122, and 124, and the CDRs of a LCVRcomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 10, 26, 42, 58, 74, 82, 123, and 125; and (b) a secondantigen-binding domain that specifically binds human CD3. In some cases,the first antigen-binding domain comprises the CDRs from a HCVR/LCVRamino acid sequence pair selected from the group consisting of SEQ IDNOs: 2/10, 18/26, 34/42, 50/58, 66/74, 122/123, 124/125, 2/82, 18/82,34/82, 50/82, 66/82, 122/82, and 124/82. In some cases, the firstantigen-binding domain comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3domains, respectively, selected from the group consisting of SEQ ID NOs:4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64,68-70-72-76-78-80, 4-6-8-84-86-88, 20-22-24-84-86-88, 36-38-40-84-86-88,52-54-56-84-86-88, and 68-70-72-84-86-88. In some cases, the firstantigen-binding domain comprises the a HCVR/LCVR amino acid sequencepair selected from the group consisting of SEQ ID NOs: 2/10, 18/26,34/42, 50/58, 66/74, 122/123, 124/125, 2/82, 18/82, 34/82, 50/82, 66/82,122/82, and 124/82. In some cases, the second antigen-binding domaincomprises the CDRs of a HCVR/LCVR amino acid sequence pair selected fromthe group consisting of SEQ ID NOs: 90/82 and 98/82.

In certain exemplary embodiments, the isolated bispecific antigenbinding molecule competes for binding to BCMA, or binds to the sameepitope on BCMA as a reference antibody, wherein the reference antibodycomprises a first antigen-binding domain comprising an HCVR/LCVR paircomprising the amino acid sequences of SEQ ID NOs: 66/82 and a secondantigen-binding domain comprising an HCVR/LCVR pair comprising the aminoacid sequences of either SEQ ID NOs: 90/82 or SEQ ID NOs: 98/82.

In certain exemplary embodiments, the isolated bispecific antigenbinding molecule competes for binding to human CD3, or binds to the sameepitope on human CD3 as a reference antibody, wherein the referenceantibody comprises a first antigen-binding domain comprising anHCVR/LCVR pair comprising the amino acid sequences of SEQ ID NOs: 66/82and a second antigen-binding domain comprising an HCVR/LCVR paircomprising the amino acid sequences of either SEQ ID NOs: 90/82 or SEQID NOs: 98/82.

The bispecific antigen-binding molecules discussed above or herein maybe bispecific antibodies. In some cases, the bispecific antibodycomprises a human IgG heavy chain constant region. In some cases, thehuman IgG heavy chain constant region is isotype IgG1. In some cases,the human IgG heavy chain constant region is isotype IgG4. In variousembodiments, the bispecific antibody comprises a chimeric hinge thatreduces Fcγ receptor binding relative to a wild-type hinge of the sameisotype.

The first antigen-binding domain and the second antigen-binding domainmay be directly or indirectly connected to one another to form abispecific antigen-binding molecule of the present invention.Alternatively, the first antigen-binding domain and the secondantigen-binding domain may each be connected to a separate multimerizingdomain. The association of one multimerizing domain with anothermultimerizing domain facilitates the association between the twoantigen-binding domains, thereby forming a bispecific antigen-bindingmolecule. As used herein, a “multimerizing domain” is any macromolecule,protein, polypeptide, peptide, or amino acid that has the ability toassociate with a second multimerizing domain of the same or similarstructure or constitution. For example, a multimerizing domain may be apolypeptide comprising an immunoglobulin C_(H)3 domain. A non-limitingexample of a multimerizing component is an Fc portion of animmunoglobulin (comprising a C_(H)2-C_(H)3 domain), e.g., an Fc domainof an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as wellas any allotype within each isotype group.

Bispecific antigen-binding molecules of the present invention willtypically comprise two multimerizing domains, e.g., two Fc domains thatare each individually part of a separate antibody heavy chain. The firstand second multimerizing domains may be of the same IgG isotype such as,e.g., IgG1/IgG1, IgG2/IgG2, IgG4/IgG4. Alternatively, the first andsecond multimerizing domains may be of different IgG isotypes such as,e.g., IgG1/IgG2, IgG1/IgG4, IgG2/IgG4, etc.

In certain embodiments, the multimerizing domain is an Fc fragment or anamino acid sequence of from 1 to about 200 amino acids in lengthcontaining at least one cysteine residue. In other embodiments, themultimerizing domain is a cysteine residue, or a shortcysteine-containing peptide. Other multimerizing domains includepeptides or polypeptides comprising or consisting of a leucine zipper, ahelix-loop motif, or a coiled-coil motif.

Any bispecific antibody format or technology may be used to make thebispecific antigen-binding molecules of the present invention. Forexample, an antibody or fragment thereof having a first antigen bindingspecificity can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmenthaving a second antigen-binding specificity to produce a bispecificantigen-binding molecule. Specific exemplary bispecific formats that canbe used in the context of the present invention include, withoutlimitation, e.g., scFv-based or diabody bispecific formats, IgG-scFvfusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes,common light chain (e.g., common light chain with knobs-into-holes,etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody,IgG1/IgG2, dual acting Fab (DAF)-IgG, and Mab² bispecific formats (see,e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein,for a review of the foregoing formats).

In the context of bispecific antigen-binding molecules of the presentinvention, the multimerizing domains, e.g., Fc domains, may comprise oneor more amino acid changes (e.g., insertions, deletions orsubstitutions) as compared to the wild-type, naturally occurring versionof the Fc domain. For example, the invention includes bispecificantigen-binding molecules comprising one or more modifications in the Fcdomain that results in a modified Fc domain having a modified bindinginteraction (e.g., enhanced or diminished) between Fc and FcRn. In oneembodiment, the bispecific antigen-binding molecule comprises amodification in a C_(H)2 or a C_(H)3 region, wherein the modificationincreases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0).Non-limiting examples of such Fc modifications include, e.g., amodification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F);252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256 (e.g., S/R/Q/E/Dor T); or a modification at position 428 and/or 433 (e.g., L/R/S/P/Q orK) and/or 434 (e.g., H/F or Y); or a modification at position 250 and/or428; or a modification at position 307 or 308 (e.g., 308F, V308F), and434. In one embodiment, the modification comprises a 428L (e.g., M428L)and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g.,434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E)modification; a 250Q and 428L modification (e.g., T250Q and M428L); anda 307 and/or 308 modification (e.g., 308F or 308P).

The present invention also includes bispecific antigen-binding moleculescomprising a first C_(H)3 domain and a second Ig C_(H)3 domain, whereinthe first and second Ig C_(H)3 domains differ from one another by atleast one amino acid, and wherein at least one amino acid differencereduces binding of the bispecific antibody to Protein A as compared to abi-specific antibody lacking the amino acid difference. In oneembodiment, the first Ig C_(H)3 domain binds Protein A and the second IgC_(H)3 domain contains a mutation that reduces or abolishes Protein Abinding such as an H95R modification (by IMGT exon numbering; H435R byEU numbering). The second C_(H)3 may further comprise a Y96Fmodification (by IMGT; Y436F by EU). See, for example, U.S. Pat. No.8,586,713. Further modifications that may be found within the secondC_(H)3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E,L358M, N384S, K392N, V397M, and V422I by EU) in the case of IgG1antibodies; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by EU)in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q,and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422Iby EU) in the case of IgG4 antibodies.

In certain embodiments, the Fc domain may be chimeric, combining Fcsequences derived from more than one immunoglobulin isotype. Forexample, a chimeric Fc domain can comprise part or all of a C_(H)2sequence derived from a human IgG1, human IgG2 or human IgG4 C_(H)2region, and part or all of a C_(H)3 sequence derived from a human IgG1,human IgG2 or human IgG4. A chimeric Fc domain can also contain achimeric hinge region. For example, a chimeric hinge may comprise an“upper hinge” sequence, derived from a human IgG1, a human IgG2 or ahuman IgG4 hinge region, combined with a “lower hinge” sequence, derivedfrom a human IgG1, a human IgG2 or a human IgG4 hinge region. Aparticular example of a chimeric Fc domain that can be included in anyof the antigen-binding molecules set forth herein comprises, from N- toC-terminus: [IgG4 C_(H)1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that can beincluded in any of the antigen-binding molecules set forth hereincomprises, from N- to C-terminus: [IgG1 C_(H)1]-[IgG1 upper hinge]-[IgG2lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimericFc domains that can be included in any of the antigen-binding moleculesof the present invention are described in US Publication 2014/0243504,published Aug. 28, 2014, which is herein incorporated in its entirety.Chimeric Fc domains having these general structural arrangements, andvariants thereof, can have altered Fc receptor binding, which in turnaffects Fc effector function.

Sequence Variants

The antibodies and bispecific antigen-binding molecules of the presentinvention may comprise one or more amino acid substitutions, insertionsand/or deletions in the framework and/or CDR regions of the heavy andlight chain variable domains as compared to the corresponding germlinesequences from which the individual antigen-binding domains werederived. Such mutations can be readily ascertained by comparing theamino acid sequences disclosed herein to germline sequences availablefrom, for example, public antibody sequence databases. Theantigen-binding molecules of the present invention may compriseantigen-binding domains which are derived from any of the exemplaryamino acid sequences disclosed herein, wherein one or more amino acidswithin one or more framework and/or CDR regions are mutated to thecorresponding residue(s) of the germline sequence from which theantibody was derived, or to the corresponding residue(s) of anotherhuman germline sequence, or to a conservative amino acid substitution ofthe corresponding germline residue(s) (such sequence changes arereferred to herein collectively as “germline mutations”). A person ofordinary skill in the art, starting with the heavy and light chainvariable region sequences disclosed herein, can easily produce numerousantibodies and antigen-binding fragments which comprise one or moreindividual germline mutations or combinations thereof. In certainembodiments, all of the framework and/or CDR residues within the V_(H)and/or V_(L) domains are mutated back to the residues found in theoriginal germline sequence from which the antigen-binding domain wasoriginally derived. In other embodiments, only certain residues aremutated back to the original germline sequence, e.g., only the mutatedresidues found within the first 8 amino acids of FR1 or within the last8 amino acids of FR4, or only the mutated residues found within CDR1,CDR2 or CDR3. In other embodiments, one or more of the framework and/orCDR residue(s) are mutated to the corresponding residue(s) of adifferent germline sequence (i.e., a germline sequence that is differentfrom the germline sequence from which the antigen-binding domain wasoriginally derived). Furthermore, the antigen-binding domains maycontain any combination of two or more germline mutations within theframework and/or CDR regions, e.g., wherein certain individual residuesare mutated to the corresponding residue of a particular germlinesequence while certain other residues that differ from the originalgermline sequence are maintained or are mutated to the correspondingresidue of a different germline sequence. Once obtained, antigen-bindingdomains that contain one or more germline mutations can be easily testedfor one or more desired property such as, improved binding specificity,increased binding affinity, improved or enhanced antagonistic oragonistic biological properties (as the case may be), reducedimmunogenicity, etc. Bispecific antigen-binding molecules comprising oneor more antigen-binding domains obtained in this general manner areencompassed within the present invention.

pH-Dependent Binding

The present invention includes anti-BCMA antibodies, and anti-BCMA xanti-CD3 bispecific antigen-binding molecules, with pH-dependent bindingcharacteristics. For example, an anti-BCMA antibody of the presentinvention may exhibit reduced binding to BCMA at acidic pH as comparedto neutral pH. Alternatively, anti-BCMA antibodies of the invention mayexhibit enhanced binding to BCMA at acidic pH as compared to neutral pH.The expression “acidic pH” includes pH values less than about 6.2, e.g.,about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45,5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As usedherein, the expression “neutral pH” means a pH of about 7.0 to about7.4. The expression “neutral pH” includes pH values of about 7.0, 7.05,7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

In certain instances, “reduced binding . . . at acidic pH as compared toneutral pH” is expressed in terms of a ratio of the K_(D) value of theantibody binding to its antigen at acidic pH to the K_(D) value of theantibody binding to its antigen at neutral pH (or vice versa). Forexample, an antibody or antigen-binding fragment thereof may be regardedas exhibiting “reduced binding to BCMA at acidic pH as compared toneutral pH” for purposes of the present invention if the antibody orantigen-binding fragment thereof exhibits an acidic/neutral K_(D) ratioof about 3.0 or greater. In certain exemplary embodiments, theacidic/neutral K_(D) ratio for an antibody or antigen-binding fragmentof the present invention can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5,13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0,100.0 or greater.

Antibodies with pH-dependent binding characteristics may be obtained,e.g., by screening a population of antibodies for reduced (or enhanced)binding to a particular antigen at acidic pH as compared to neutral pH.Additionally, modifications of the antigen-binding domain at the aminoacid level may yield antibodies with pH-dependent characteristics. Forexample, by substituting one or more amino acids of an antigen-bindingdomain (e.g., within a CDR) with a histidine residue, an antibody withreduced antigen-binding at acidic pH relative to neutral pH may beobtained.

Antibodies Comprising Fc Variants

According to certain embodiments of the present invention, anti-BCMAantibodies, and anti-BCMA x anti-CD3 bispecific antigen-bindingmolecules, are provided comprising an Fc domain comprising one or moremutations which enhance or diminish antibody binding to the FcRnreceptor, e.g., at acidic pH as compared to neutral pH. For example, thepresent invention includes antibodies comprising a mutation in theC_(H)2 or a C_(H)3 region of the Fc domain, wherein the mutation(s)increases the affinity of the Fc domain to FcRn in an acidic environment(e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Suchmutations may result in an increase in serum half-life of the antibodywhen administered to an animal. Non-limiting examples of such Fcmodifications include, e.g., a modification at position 250 (e.g., E orQ); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., Sor T), and 256 (e.g., S/R/Q/E/D or T); or a modification at position 428and/or 433 (e.g., H/L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or amodification at position 250 and/or 428; or a modification at position307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, themodification comprises a 428L (e.g., M428L) and 434S (e.g., N434S)modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F)modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification;a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Qand 428L modification (e.g., T250Q and M428L); and a 307 and/or 308modification (e.g., 308F or 308P).

For example, the present invention includes anti-BCMA antibodies, andanti-BCMA x anti-CD3 bispecific antigen-binding molecules, comprising anFc domain comprising one or more pairs or groups of mutations selectedfrom the group consisting of 250Q and 248L (e.g., T250Q and M248L);252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g.,M428L and N434S); and 433K and 434F (e.g., H433K and N434F). Allpossible combinations of the foregoing Fc domain mutations, and othermutations within the antibody variable domains disclosed herein, arecontemplated within the scope of the present invention.

Biological Characteristics of the Antibodies and BispecificAntigen-Binding Molecules

The present invention includes antibodies and antigen-binding fragmentsthereof that bind human BCMA with high affinity (e.g., nanomolar orsub-nanomolar K_(D) values).

According to certain embodiments, the present invention includesantibodies and antigen-binding fragments of antibodies that bind humanBCMA (e.g., at 25° C.) with a K_(D) of less than about 5 nM as measuredby surface plasmon resonance, e.g., using an assay format as defined inExample 4 herein. In certain embodiments, the antibodies orantigen-binding fragments of the present invention bind BCMA with aK_(D) of less than about 20 nM, less than about 10 nM, less than about 8nM, less than about 7 nM, less than about 6 nM, less than about 5 nM,less than about 4 nM, less than about 3 nM, less than about 2 nM, lessthan about 1 nM, less than about 800 pM, less than about 700 pM, lessthan about 500 pM, less than about 400 pM, less than about 300 pM, lessthan about 200 pM, less than about 100 pM, less than about 50 pM, orless than about 25 pM as measured by surface plasmon resonance, e.g.,using an assay format as defined in Example 4 herein, or a substantiallysimilar assay. The present invention includes bispecific antigen-bindingmolecules (e.g., bispecific antibodies which bind human BCMA with aK_(D) of less than about 25 pM, and which bind monkey BCMA with a K_(D)of less than about 170 pM, as measured by surface plasmon resonance,e.g., using an assay format as defined in Example 4 herein, or asubstantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof that bind BCMA with a dissociative half-life (t %) ofgreater than about 10 minutes or greater than about 125 minutes asmeasured by surface plasmon resonance at 25° C., e.g., using an assayformat as defined in Example 4 herein, or a substantially similar assay.In certain embodiments, the antibodies or antigen-binding fragments ofthe present invention bind BCMA with a t % of greater than about 3minutes, greater than about 4 minutes, greater than about 10 minutes,greater than about 20 minutes, greater than about 30 minutes, greaterthan about 40 minutes, greater than about 50 minutes, greater than about60 minutes, greater than about 70 minutes, greater than about 80minutes, greater than about 90 minutes, greater than about 100 minutes,greater than about 110 minutes, or greater than about 120 minutes, asmeasured by surface plasmon resonance at 25° C., e.g., using an assayformat as defined in Example 4 herein, or a substantially similar assay.The present invention includes bispecific antigen-binding molecules(e.g., bispecific antibodies which bind BCMA with a of greater thanabout 10 minutes as measured by surface plasmon resonance at 25° C.,e.g., using an assay format as defined in Example 4 herein, or asubstantially similar assay.

The present invention also includes antibodies and antigen-bindingfragments thereof which bind specifically to human cell lines whichexpress endogenous BCMA (e.g., NCI-H929, MOLP-8 or OMP-2), as determinedby a FACS binding assay as set forth in Example 6 or a substantiallysimilar assay.

The present invention also includes anti-BCMA x anti-CD3 bispecificantigen-binding molecules which exhibit one or more characteristicsselected from the group consisting of (a) inhibiting tumor growth inimmunocompromised mice bearing human multiple myeloma xenografts; (b)suppressing tumor growth of established tumors in immunocompromised micebearing human multiple myeloma xenografts (see, e.g., Examples 10-15),and (c) suppressing tumor growth of syngenic melanoma and coloncarcinoma cells engineered to express human BCMA in immunocompetent miceexpressing human CD3.

The present invention includes antibodies and antigen-binding fragmentsthereof that bind human CD3 with high affinity. The present inventionalso includes antibodies and antigen-binding fragments thereof that bindhuman CD3 with medium or low affinity, depending on the therapeuticcontext and particular targeting properties that are desired. In somecases, the low affinity includes antibodies that bind CD3 with a K_(D)or EC₅₀ (e.g., as measured in a surface plasmon resonance assay) ofgreater than 300 nM, greater than 500 nM or greater than 1 pM. Thepresent invention also includes antibodies and antigen-binding fragmentsthereof that bind human CD3 with no measurable affinity. For example, inthe context of a bispecific antigen-binding molecule, wherein one armbinds CD3 and another arm binds a target antigen (e.g., BCMA), it may bedesirable for the target antigen-binding arm to bind the target antigenwith high affinity while the anti-CD3 arm binds CD3 with only moderateor low affinity or no affinity. In this manner, preferential targetingof the antigen-binding molecule to cells expressing the target antigenmay be achieved while avoiding general/untargeted CD3 binding and theconsequent adverse side effects associated therewith.

The present invention includes bispecific antigen-binding molecules(e.g., bispecific antibodies) which are capable of simultaneouslybinding to human CD3 and a human BCMA. The binding arm that interactswith cells that express CD3 may have weak to no detectable binding asmeasured in a suitable in vitro binding assay. The extent to which abispecific antigen-binding molecule binds cells that express CD3 and/orBCMA can be assessed by fluorescence activated cell sorting (FACS), asillustrated in Examples 5 and 6 herein.

For example, the present invention includes antibodies, antigen-bindingfragments, and bispecific antibodies thereof which specifically bindhuman T-cell lines which express CD3 but do not express BCMA (e.g.,Jurkat), and/or BCMA-expressing cells.

The present invention includes antibodies, antigen-binding fragments,and bispecific antibodies thereof that bind human CD3 with weak (i.e.low) or even no detectable affinity.

The present invention includes antibodies, antigen-binding fragments,and bispecific antibodies thereof that bind monkey (i.e. cynomolgus) CD3with weak (i.e. low) or even no detectable affinity.

The present invention includes antibodies, antigen-binding fragments,and bispecific antibodies thereof that bind human CD3 and induce T cellactivation.

The present invention includes anti-BCMA x anti-CD3 bispecificantigen-binding molecules which are capable of depleting or reducingtumor antigen-expressing cells in a subject (see, e.g., Examples 8-16,or a substantially similar assay). For example, according to certainembodiments, anti-BCMA x anti-CD3 bispecific antigen-binding moleculesare provided, wherein a single administration, or multipleadministrations, of 0.04 mg/kg, 0.4 mg/kg or 4 mg/kg of the bispecificantigen-binding molecule to a subject causes a reduction in the numberof BCMA-expressing cells in the subject (e.g., tumor growth in thesubject is suppressed or inhibited).

Epitope Mapping and Related Technologies

The epitope on CD3 and/or BCMA to which the antigen-binding molecules ofthe present invention bind may consist of a single contiguous sequenceof 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more) amino acids of a CD3 or BCMA protein. Alternatively,the epitope may consist of a plurality of non-contiguous amino acids (oramino acid sequences) of CD3 or BCMA. The antibodies of the inventionmay interact with amino acids contained within a single CD3 chain (e.g.,CD3-epsilon, CD3-delta or CD3-gamma), or may interact with amino acidson two or more different CD3 chains. The term “epitope,” as used herein,refers to an antigenic determinant that interacts with a specificantigen binding site in the variable region of an antibody moleculeknown as a paratope. A single antigen may have more than one epitope.Thus, different antibodies may bind to different areas on an antigen andmay have different biological effects. Epitopes may be eitherconformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. In certain circumstances, anepitope may include moieties of saccharides, phosphoryl groups, orsulfonyl groups on the antigen.

Various techniques known to persons of ordinary skill in the art can beused to determine whether an antigen-binding domain of an antibody“interacts with one or more amino acids” within a polypeptide orprotein. Exemplary techniques include, e.g., routine cross-blockingassay such as that described Antibodies. Harlow and Lane (Cold SpringHarbor Press, Cold Spring Harb., NY), alanine scanning mutationalanalysis, peptide blots analysis (Reineke, 2004, Methods Mol Biol248:443-463), and peptide cleavage analysis. In addition, methods suchas epitope excision, epitope extraction and chemical modification ofantigens can be employed (Tomer, 2000, Protein Science 9:487-496).Another method that can be used to identify the amino acids within apolypeptide with which an antigen-binding domain of an antibodyinteracts is hydrogen/deuterium exchange detected by mass spectrometry.In general terms, the hydrogen/deuterium exchange method involvesdeuterium-labeling the protein of interest, followed by binding theantibody to the deuterium-labeled protein. Next, the protein/antibodycomplex is transferred to water to allow hydrogen-deuterium exchange tooccur at all residues except for the residues protected by the antibody(which remain deuterium-labeled). After dissociation of the antibody,the target protein is subjected to protease cleavage and massspectrometry analysis, thereby revealing the deuterium-labeled residueswhich correspond to the specific amino acids with which the antibodyinteracts. See, e.g., Ehring (1999) Analytical Biochemistry267(2):252-259; Engen and Smith (2001) Anal. Chem. 73.256A-265A. X-raycrystallography of the antigen/antibody complex may also be used forepitope mapping purposes.

The present invention further includes anti-BCMA antibodies that bind tothe same epitope as any of the specific exemplary antibodies describedherein (e.g. antibodies comprising any of the amino acid sequences asset forth in Table 1 herein). Likewise, the present invention alsoincludes anti-BCMA antibodies that compete for binding to BCMA with anyof the specific exemplary antibodies described herein (e.g. antibodiescomprising any of the amino acid sequences as set forth in Table 1herein).

The present invention also includes bispecific antigen-binding moleculescomprising a second antigen-binding domain that specifically binds humanCD3 and/or cynomolgus CD3 with low or no detectable binding affinity,and a second antigen binding domain that specifically binds human BCMA,wherein the second antigen-binding domain binds to the same epitope onCD3 as any of the specific exemplary CD3-specific antigen-bindingdomains described herein, and/or wherein the second antigen-bindingdomain binds to the same epitope on BCMA as any of the specificexemplary BCMA-specific antigen-binding domains described herein.

Likewise, the present invention also includes bispecific antigen-bindingmolecules comprising a first antigen-binding domain that specificallybinds human BCMA, and a second antigen binding domain that specificallybinds human CD3, wherein the first antigen-binding domain competes forbinding to BCMA with any of the specific exemplary BCMA-specificantigen-binding domains described herein, and/or wherein the secondantigen-binding domain competes for binding to CD3 with any of thespecific exemplary CD3-specific antigen-binding domains describedherein.

One can easily determine whether a particular antigen-binding molecule(e.g., antibody) or antigen-binding domain thereof binds to the sameepitope as, or competes for binding with, a reference antigen-bindingmolecule of the present invention by using routine methods known in theart. For example, to determine if a test antibody binds to the sameepitope on BCMA (or CD3) as a reference bispecific antigen-bindingmolecule of the present invention, the reference bispecific molecule isfirst allowed to bind to a BCMA protein (or CD3 protein). Next, theability of a test antibody to bind to the BCMA (or CD3) molecule isassessed. If the test antibody is able to bind to BCMA (or CD3)following saturation binding with the reference bispecificantigen-binding molecule, it can be concluded that the test antibodybinds to a different epitope of BCMA (or CD3) than the referencebispecific antigen-binding molecule. On the other hand, if the testantibody is not able to bind to the BCMA (or CD3) molecule followingsaturation binding with the reference bispecific antigen-bindingmolecule, then the test antibody may bind to the same epitope of BCMA(or CD3) as the epitope bound by the reference bispecificantigen-binding molecule of the invention. Additional routineexperimentation (e.g., peptide mutation and binding analyses) can thenbe carried out to confirm whether the observed lack of binding of thetest antibody is in fact due to binding to the same epitope as thereference bispecific antigen-binding molecule or if steric blocking (oranother phenomenon) is responsible for the lack of observed binding.Experiments of this sort can be performed using ELISA, RIA, Biacore,flow cytometry or any other quantitative or qualitative antibody-bindingassay available in the art. In accordance with certain embodiments ofthe present invention, two antigen-binding proteins bind to the same (oroverlapping) epitope if, e.g., a 1-, 5-, 10-, 20- or 100-fold excess ofone antigen-binding protein inhibits binding of the other by at least50% but preferably 75%, 90% or even 99% as measured in a competitivebinding assay (see, e.g., Junghans et al., Cancer Res.1990:50:1495-1502). Alternatively, two antigen-binding proteins aredeemed to bind to the same epitope if essentially all amino acidmutations in the antigen that reduce or eliminate binding of oneantigen-binding protein reduce or eliminate binding of the other. Twoantigen-binding proteins are deemed to have “overlapping epitopes” ifonly a subset of the amino acid mutations that reduce or eliminatebinding of one antigen-binding protein reduce or eliminate binding ofthe other.

To determine if an antibody or antigen-binding domain thereof competesfor binding with a reference antigen-binding molecule, theabove-described binding methodology is performed in two orientations: Ina first orientation, the reference antigen-binding molecule is allowedto bind to a BCMA protein (or CD3 protein) under saturating conditionsfollowed by assessment of binding of the test antibody to the BCMA (orCD3) molecule. In a second orientation, the test antibody is allowed tobind to a BCMA (or CD3) molecule under saturating conditions followed byassessment of binding of the reference antigen-binding molecule to theBCMA (or CD3) molecule. If, in both orientations, only the first(saturating) antigen-binding molecule is capable of binding to the BCMA(or CD3) molecule, then it is concluded that the test antibody and thereference antigen-binding molecule compete for binding to BCMA (or CD3).As will be appreciated by a person of ordinary skill in the art, anantibody that competes for binding with a reference antigen-bindingmolecule may not necessarily bind to the same epitope as the referenceantibody, but may sterically block binding of the reference antibody bybinding an overlapping or adjacent epitope.

Preparation of Antigen-Binding Domains and Construction of BispecificMolecules

Antigen-binding domains specific for particular antigens can be preparedby any antibody generating technology known in the art. Once obtained,two different antigen-binding domains, specific for two differentantigens (e.g., CD3 and BCMA), can be appropriately arranged relative toone another to produce a bispecific antigen-binding molecule of thepresent invention using routine methods. (A discussion of exemplarybispecific antibody formats that can be used to construct the bispecificantigen-binding molecules of the present invention is provided elsewhereherein). In certain embodiments, one or more of the individualcomponents (e.g., heavy and light chains) of the multispecificantigen-binding molecules of the invention are derived from chimeric,humanized or fully human antibodies. Methods for making such antibodiesare well known in the art. For example, one or more of the heavy and/orlight chains of the bispecific antigen-binding molecules of the presentinvention can be prepared using VELOCIMMUNE™ technology. UsingVELOCIMMUNE™ technology (or any other human antibody generatingtechnology), high affinity chimeric antibodies to a particular antigen(e.g., CD3 or BCMA) are initially isolated having a human variableregion and a mouse constant region. The antibodies are characterized andselected for desirable characteristics, including affinity, selectivity,epitope, etc. The mouse constant regions are replaced with a desiredhuman constant region to generate fully human heavy and/or light chainsthat can be incorporated into the bispecific antigen-binding moleculesof the present invention.

Genetically engineered animals may be used to make human bispecificantigen-binding molecules. For example, a genetically modified mouse canbe used which is incapable of rearranging and expressing an endogenousmouse immunoglobulin light chain variable sequence, wherein the mouseexpresses only one or two human light chain variable domains encoded byhuman immunoglobulin sequences operably linked to the mouse kappaconstant gene at the endogenous mouse kappa locus. Such geneticallymodified mice can be used to produce fully human bispecificantigen-binding molecules comprising two different heavy chains thatassociate with an identical light chain that comprises a variable domainderived from one of two different human light chain variable region genesegments. (See, e.g., US 2011/0195454). Fully human refers to anantibody, or antigen-binding fragment or immunoglobulin domain thereof,comprising an amino acid sequence encoded by a DNA derived from a humansequence over the entire length of each polypeptide of the antibody orantigen-binding fragment or immunoglobulin domain thereof. In someinstances, the fully human sequence is derived from a protein endogenousto a human. In other instances, the fully human protein or proteinsequence comprises a chimeric sequence wherein each component sequenceis derived from human sequence. While not being bound by any one theory,chimeric proteins or chimeric sequences are generally designed tominimize the creation of immunogenic epitopes in the junctions ofcomponent sequences, e.g. compared to any wild-type human immunoglobulinregions or domains.

Bioequivalents

The present invention encompasses antigen-binding molecules having aminoacid sequences that vary from those of the exemplary molecules disclosedherein but that retain the ability to bind CD3 and/or BCMA. Such variantmolecules may comprise one or more additions, deletions, orsubstitutions of amino acids when compared to parent sequence, butexhibit biological activity that is essentially equivalent to that ofthe described bispecific antigen-binding molecules.

The present invention includes antigen-binding molecules that arebioequivalent to any of the exemplary antigen-binding molecules setforth herein. Two antigen-binding proteins, or antibodies, areconsidered bioequivalent if, for example, they are pharmaceuticalequivalents or pharmaceutical alternatives whose rate and extent ofabsorption do not show a significant difference when administered at thesame molar dose under similar experimental conditions, either singledoes or multiple dose. Some antigen-binding proteins will be consideredequivalents or pharmaceutical alternatives if they are equivalent in theextent of their absorption but not in their rate of absorption and yetmay be considered bioequivalent because such differences in the rate ofabsorption are intentional and are reflected in the labeling, are notessential to the attainment of effective body drug concentrations on,e.g., chronic use, and are considered medically insignificant for theparticular drug product studied.

In one embodiment, two antigen-binding proteins are bioequivalent ifthere are no clinically meaningful differences in their safety, purity,and potency.

In one embodiment, two antigen-binding proteins are bioequivalent if apatient can be switched one or more times between the reference productand the biological product without an expected increase in the risk ofadverse effects, including a clinically significant change inimmunogenicity, or diminished effectiveness, as compared to continuedtherapy without such switching.

In one embodiment, two antigen-binding proteins are bioequivalent ifthey both act by a common mechanism or mechanisms of action for thecondition or conditions of use, to the extent that such mechanisms areknown.

Bioequivalence may be demonstrated by in vivo and in vitro methods.Bioequivalence measures include, e.g., (a) an in vivo test in humans orother mammals, in which the concentration of the antibody or itsmetabolites is measured in blood, plasma, serum, or other biologicalfluid as a function of time; (b) an in vitro test that has beencorrelated with and is reasonably predictive of human in vivobioavailability data; (c) an in vivo test in humans or other mammals inwhich the appropriate acute pharmacological effect of the antibody (orits target) is measured as a function of time; and (d) in awell-controlled clinical trial that establishes safety, efficacy, orbioavailability or bioequivalence of an antigen-binding protein.

Bioequivalent variants of the exemplary bispecific antigen-bindingmolecules set forth herein may be constructed by, for example, makingvarious substitutions of residues or sequences or deleting terminal orinternal residues or sequences not needed for biological activity. Forexample, cysteine residues not essential for biological activity can bedeleted or replaced with other amino acids to prevent formation ofunnecessary or incorrect intramolecular disulfide bridges uponrenaturation. In other contexts, bioequivalent antigen-binding proteinsmay include variants of the exemplary bispecific antigen-bindingmolecules set forth herein comprising amino acid changes which modifythe glycosylation characteristics of the molecules, e.g., mutationswhich eliminate or remove glycosylation.

Species Selectivity and Species Cross-Reactivity

According to certain embodiments of the invention, antigen-bindingmolecules are provided which bind to human CD3 but not to CD3 from otherspecies. Also provided are antigen-binding molecules which bind to humanBCMA, but not to BCMA from other species. The present invention alsoincludes antigen-binding molecules that bind to human CD3 and to CD3from one or more non-human species; and/or antigen-binding moleculesthat bind to human BCMA and to BCMA from one or more non-human species.

According to certain exemplary embodiments of the invention,antigen-binding molecules are provided which bind to human CD3 and/orhuman BCMA and may bind or not bind, as the case may be, to one or moreof mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat,sheep, cow, horse, camel, cynomolgus, marmoset, rhesus or chimpanzee CD3and/or BCMA. For example, in particular exemplary embodiments of thepresent invention bispecific antigen-binding molecules are providedcomprising a first antigen-binding domain that binds human BCMA andcynomolgus BCMA, and a second antigen-binding domain that specificallybinds human CD3, or bispecific antigen-binding molecules comprising afirst antigen-binding domain that binds human BCMA and cynomolgus BCMA,and a second antigen-binding domain that specifically binds human CD3.

Therapeutic Formulation and Administration

The present invention provides pharmaceutical compositions comprisingthe antigen-binding molecules of the present invention. Thepharmaceutical compositions of the invention are formulated withsuitable carriers, excipients, and other agents that provide improvedtransfer, delivery, tolerance, and the like. A multitude of appropriateformulations can be found in the formulary known to all pharmaceuticalchemists: Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa. These formulations include, for example, powders, pastes,ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad,Calif.), DNA conjugates, anhydrous absorption pastes, oil-in-water andwater-in-oil emulsions, emulsions carbowax (polyethylene glycols ofvarious molecular weights), semi-solid gels, and semi-solid mixturescontaining carbowax. See also Powell et al. “Compendium of excipientsfor parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

The dose of antigen-binding molecule administered to a patient may varydepending upon the age and the size of the patient, target disease,conditions, route of administration, and the like. The preferred dose istypically calculated according to body weight or body surface area. Whena bispecific antigen-binding molecule of the present invention is usedfor therapeutic purposes in an adult patient, it may be advantageous tointravenously administer the bispecific antigen-binding molecule of thepresent invention normally at a single dose of about 0.01 to about 20mg/kg body weight, more preferably about 0.02 to about 7, about 0.03 toabout 5, or about 0.05 to about 3 mg/kg body weight. Depending on theseverity of the condition, the frequency and the duration of thetreatment can be adjusted. Effective dosages and schedules foradministering a bispecific antigen-binding molecule may be determinedempirically; for example, patient progress can be monitored by periodicassessment, and the dose adjusted accordingly. Moreover, interspeciesscaling of dosages can be performed using well-known methods in the art(e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

A pharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present invention. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(sanofi-aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park Ill.), to name only a few.

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla. In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson,1984, in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138). Other controlled release systems are discussed in the reviewby Langer, 1990, Science 249:1527-1533.

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule.

Advantageously, the pharmaceutical compositions for oral or parenteraluse described above are prepared into dosage forms in a unit dose suitedto fit a dose of the active ingredients. Such dosage forms in a unitdose include, for example, tablets, pills, capsules, injections(ampoules), suppositories, etc. The amount of the aforesaid antibodycontained is generally about 5 to about 500 mg per dosage form in a unitdose; especially in the form of injection, it is preferred that theaforesaid antibody is contained in about 5 to about 100 mg and in about10 to about 250 mg for the other dosage forms.

Therapeutic Uses of the Antigen-Binding Molecules

The present invention includes methods comprising administering to asubject in need thereof a therapeutic composition comprising ananti-BCMA antibody or antigen-binding fragment thereof, or a bispecificantigen-binding molecule that specifically binds CD3 and BCMA. Thetherapeutic composition can comprise any of the antibodies or bispecificantigen-binding molecules as disclosed herein and a pharmaceuticallyacceptable carrier or diluent. As used herein, the expression “a subjectin need thereof” means a human or non-human animal that exhibits one ormore symptoms or indicia of cancer (e.g., a subject expressing a tumoror suffering from any of the cancers mentioned herein below), or whootherwise would benefit from an inhibition or reduction in BCMA activityor a depletion of BCMA+ cells (e.g., multiple myeloma cells).

The antibodies and bispecific antigen-binding molecules of the invention(and therapeutic compositions comprising the same) are useful, interalia, for treating any disease or disorder in which stimulation,activation and/or targeting of an immune response would be beneficial.In particular, the anti-BCMA antibodies or the anti-BCMA x anti-CD3bispecific antigen-binding molecules of the present invention may beused for the treatment, prevention and/or amelioration of any disease ordisorder associated with or mediated by BCMA expression or activity orthe proliferation of BCMA+ cells. The mechanism of action by which thetherapeutic methods of the invention are achieved include killing of thecells expressing BCMA in the presence of effector cells, for example, byCDC, apoptosis, ADCC, phagocytosis, or by a combination of two or moreof these mechanisms. Cells expressing BCMA which can be inhibited orkilled using the bispecific antigen-binding molecules of the inventioninclude, for example, multiple myeloma cells.

The antigen-binding molecules of the present invention may be used totreat a disease or disorder associates with BCMA expression including,e.g., a cancer including multiple myeloma or other B-cell or plasma cellcancers, such as Waldenström's macroglobulinemia, Burkitt lymphoma, anddiffuse large B-Cell lymphoma, Non-Hodgkin's lymphoma, chroniclymphocytic leukemia, follicular lymphoma, mantle cell lymphoma,marginal zone lymphoma, lymphoplasmacytic lymphoma, and Hodgkin'slymphoma. According to certain embodiments of the present invention, theanti-BCMA antibodies or anti-BCMA x anti-CD3 bispecific antibodies areuseful for treating a patient afflicted with multiple myeloma. Accordingto other related embodiments of the invention, methods are providedcomprising administering an anti-BCMA antibody or an anti-BCMA xanti-CD3 bispecific antigen-binding molecule as disclosed herein to apatient who is afflicted with multiple myeloma. Analytic/diagnosticmethods known in the art, such as tumor scanning, etc., may be used toascertain whether a patient harbors multiple myeloma or another B-celllineage cancer.

The present invention also includes methods for treating residual cancerin a subject. As used herein, the term “residual cancer” means theexistence or persistence of one or more cancerous cells in a subjectfollowing treatment with an anti-cancer therapy.

According to certain aspects, the present invention provides methods fortreating a disease or disorder associated with BCMA expression (e.g.,multiple myeloma) comprising administering one or more of the anti-BCMAor bispecific antigen-binding molecules described elsewhere herein to asubject after the subject has been determined to have multiple myeloma.For example, the present invention includes methods for treatingmultiple myeloma comprising administering an anti-BCMA antibody or ananti-BCMA x anti-CD3 bispecific antigen-binding molecule to a patient 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or4 weeks, 2 months, 4 months, 6 months, 8 months, 1 year, or more afterthe subject has received other immunotherapy or chemotherapy.

Combination Therapies and Formulations

The present invention provides methods which comprise administering apharmaceutical composition comprising any of the exemplary antibodiesand bispecific antigen-binding molecules described herein in combinationwith one or more additional therapeutic agents. Exemplary additionaltherapeutic agents that may be combined with or administered incombination with an antigen-binding molecule of the present inventioninclude, e.g., an anti-tumor agent (e.g. chemotherapeutic agentsincluding melphalan, vincristine (Oncovin), cyclophosphamide (Cytoxan),etoposide (VP-16), doxorubicin (Adriamycin), liposomal doxorubicin(Doxil), obendamustine (Treanda), or any others known to be effective intreating a plasma cell tumor in a subject). In some embodiments, thesecond therapeutic agent comprises steroids. In some embodiments, thesecond therapeutic agent comprises targeted therapies includingthalidomide, lenalidomide, and bortezomib, which are therapies approvedto treat newly diagnosed patients. Lenalidomide, pomalidomide,bortezomib, carfilzomib, panobinostat, ixazomib, elotuzumab, anddaratumumab are examples of a second therapeutic agent effective fortreating recurrent myeloma. In certain embodiments the secondtherapeutic agent is a regimen comprising radiotherapy or a stem celltransplant. In certain embodiments, the second therapeutic agent may bean immunomodulatory agent. In certain embodiments, the secondtherapeutic agent may be a proteasome inhibitor, including bortezomib(Velcade), carfilzomib (Kyprolis), ixazomib (Ninlaro). In certainembodiments the second therapeutic agent may be a histone deacetylaseinhibitor such as panobinostat (Farydak). In certain embodiments, thesecond therapeutic agent may be a monoclonal antibody, an antibody drugconjugate, a bispecific antibody conjugated to an anti-tumor agent, acheckpoint inhibitor, or combinations thereof. Other agents that may bebeneficially administered in combination with the antigen-bindingmolecules of the invention include cytokine inhibitors, includingsmall-molecule cytokine inhibitors and antibodies that bind to cytokinessuch as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12,IL-13, IL-17, IL-18, or to their respective receptors. Thepharmaceutical compositions of the present invention (e.g.,pharmaceutical compositions comprising an anti-BCMA x anti-CD3bispecific antigen-binding molecule as disclosed herein) may also beadministered as part of a therapeutic regimen comprising one or moretherapeutic combinations selected from a monoclonal antibody other thanthose described herein, which may interact with a different antigen onthe plasma cell surface, a bispecific antibody, which has one arm thatbinds to an antigen on the tumor cell surface and the other arm binds toan antigen on a T cell, an antibody drug conjugate, a bispecificantibody conjugated with an anti-tumor agent, a checkpoint inhibitor,for example, one that targets, PD-1 or CTLA-4, or combinations thereof.In certain embodiments, the checkpoint inhibitors may be selected fromPD-1 inhibitors, such as pembrolizumab (Keytruda), nivolumab (Opdivo),or cemiplimab (REGN2810). In certain embodiments, the checkpointinhibitors may be selected from PD-L1 inhibitors, such as atezolizumab(Tecentriq), avelumab (Bavencio), or Durvalumab (Imfinzi)). In certainembodiments, the checkpoint inhibitors may be selected from CTLA-4inhibitors, such as ipilimumab (Yervoy). Other combinations that may beused in conjunction with an antibody of the invention are describedabove.

The present invention also includes therapeutic combinations comprisingany of the antigen-binding molecules mentioned herein and an inhibitorof one or more of VEGF, Ang2, DLL4, EGFR, ErbB2, ErbB3, ErbB4, EGFRvIII,cMet, IGF1R, B-raf, PDGFR-α, PDGFR-β, FOLH1 (PSMA), PRLR, STEAP1,STEAP2, TMPRSS2, MSLN, CA9, uroplakin, or any of the aforementionedcytokines, wherein the inhibitor is an aptamer, an antisense molecule, aribozyme, an siRNA, a peptibody, a nanobody or an antibody fragment(e.g., Fab fragment; F(ab′)₂ fragment; Fd fragment; Fv fragment; scFv;dAb fragment; or other engineered molecules, such as diabodies,triabodies, tetrabodies, minibodies and minimal recognition units). Theantigen-binding molecules of the invention may also be administeredand/or co-formulated in combination with antivirals, antibiotics,analgesics, corticosteroids and/or NSAIDs. The antigen-binding moleculesof the invention may also be administered as part of a treatment regimenthat also includes radiation treatment and/or conventional chemotherapy.

The additional therapeutically active component(s) may be administeredjust prior to, concurrent with, or shortly after the administration ofan antigen-binding molecule of the present invention; (for purposes ofthe present disclosure, such administration regimens are considered theadministration of an antigen-binding molecule “in combination with” anadditional therapeutically active component).

The present invention includes pharmaceutical compositions in which anantigen-binding molecule of the present invention is co-formulated withone or more of the additional therapeutically active component(s) asdescribed elsewhere herein.

Administration Regimens

According to certain embodiments of the present invention, multipledoses of an antigen-binding molecule (e.g., an anti-BCMA antibody or abispecific antigen-binding molecule that specifically binds BCMA andCD3) may be administered to a subject over a defined time course. Themethods according to this aspect of the invention comprise sequentiallyadministering to a subject multiple doses of an antigen-binding moleculeof the invention. As used herein, “sequentially administering” meansthat each dose of an antigen-binding molecule is administered to thesubject at a different point in time, e.g., on different days separatedby a predetermined interval (e.g., hours, days, weeks or months). Thepresent invention includes methods which comprise sequentiallyadministering to the patient a single initial dose of an antigen-bindingmolecule, followed by one or more secondary doses of the antigen-bindingmolecule, and optionally followed by one or more tertiary doses of theantigen-binding molecule.

The terms “initial dose,” “secondary doses,” and “tertiary doses,” referto the temporal sequence of administration of the antigen-bindingmolecule of the invention. Thus, the “initial dose” is the dose which isadministered at the beginning of the treatment regimen (also referred toas the “baseline dose”); the “secondary doses” are the doses which areadministered after the initial dose; and the “tertiary doses” are thedoses which are administered after the secondary doses. The initial,secondary, and tertiary doses may all contain the same amount of theantigen-binding molecule, but generally may differ from one another interms of frequency of administration. In certain embodiments, however,the amount of an antigen-binding molecule contained in the initial,secondary and/or tertiary doses varies from one another (e.g., adjustedup or down as appropriate) during the course of treatment. In certainembodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered atthe beginning of the treatment regimen as “loading doses” followed bysubsequent doses that are administered on a less frequent basis (e.g.,“maintenance doses”).

In one exemplary embodiment of the present invention, each secondaryand/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½,4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 1½, 12, 12½, 13,13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21,21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks afterthe immediately preceding dose. The phrase “the immediately precedingdose,” as used herein, means, in a sequence of multiple administrations,the dose of antigen-binding molecule which is administered to a patientprior to the administration of the very next dose in the sequence withno intervening doses.

The methods according to this aspect of the invention may compriseadministering to a patient any number of secondary and/or tertiary dosesof an antigen-binding molecule (e.g., an anti-BCMA antibody or abispecific antigen-binding molecule that specifically binds BCMA andCD3). For example, in certain embodiments, only a single secondary doseis administered to the patient. In other embodiments, two or more (e.g.,2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to thepatient. Likewise, in certain embodiments, only a single tertiary doseis administered to the patient. In other embodiments, two or more (e.g.,2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to thepatient.

In embodiments involving multiple secondary doses, each secondary dosemay be administered at the same frequency as the other secondary doses.For example, each secondary dose may be administered to the patient 1 to2 weeks after the immediately preceding dose. Similarly, in embodimentsinvolving multiple tertiary doses, each tertiary dose may beadministered at the same frequency as the other tertiary doses. Forexample, each tertiary dose may be administered to the patient 2 to 4weeks after the immediately preceding dose. Alternatively, the frequencyat which the secondary and/or tertiary doses are administered to apatient can vary over the course of the treatment regimen. The frequencyof administration may also be adjusted during the course of treatment bya physician depending on the needs of the individual patient followingclinical examination.

Diagnostic Uses of the Antibodies

The anti-BCMA antibodies of the present invention may also be used todetect and/or measure BCMA, or BCMA-expressing cells in a sample, e.g.,for diagnostic purposes. For example, an anti-BCMA antibody, or fragmentthereof, may be used to diagnose a condition or disease characterized byaberrant expression (e.g., over-expression, under-expression, lack ofexpression, etc.) of BCMA. Exemplary diagnostic assays for BCMA maycomprise, e.g., contacting a sample, obtained from a patient, with ananti-BCMA antibody of the invention, wherein the anti-BCMA antibody islabeled with a detectable label or reporter molecule. Alternatively, anunlabeled anti-BCMA antibody can be used in diagnostic applications incombination with a secondary antibody which is itself detectablylabeled. The detectable label or reporter molecule can be aradioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I; a fluorescent orchemiluminescent moiety such as fluorescein isothiocyanate, orrhodamine; or an enzyme such as alkaline phosphatase,beta-galactosidase, horseradish peroxidase, or luciferase. Anotherexemplary diagnostic use of the anti-BCMA antibodies of the inventionincludes ⁸⁹Zr-labeled, such as ⁸⁹Zr-desferrioxamine-labeled, antibodyfor the purpose of noninvasive identification and tracking of tumorcells in a subject (e.g. positron emission tomography (PET) imaging).(See, e.g., Tavare, R. et al. Cancer Res. 2016 Jan. 1; 76(1):73-82; andAzad, B B. et al. Oncotarget. 2016 Mar. 15; 7(11):12344-58.) Specificexemplary assays that can be used to detect or measure BCMA in a sampleinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence-activated cell sorting (FACS).

Samples that can be used in BCMA diagnostic assays according to thepresent invention include any tissue or fluid sample obtainable from apatient which contains detectable quantities of BCMA protein, orfragments thereof, under normal or pathological conditions. Generally,levels of BCMA in a particular sample obtained from a healthy patient(e.g., a patient not afflicted with a disease or condition associatedwith abnormal BCMA levels or activity) will be measured to initiallyestablish a baseline, or standard, level of BCMA. This baseline level ofBCMA can then be compared against the levels of BCMA measured in samplesobtained from individuals suspected of having a BCMA related disease(e.g., a tumor containing BCMA-expressing cells) or condition.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1: Generation of Anti-BCMA Antibodies

Anti-BCMA antibodies were obtained by immunizing a genetically modifiedmouse with a human BCMA antigen (e.g., hBCMA, SEQ ID NO: 115) or byimmunizing an engineered mouse comprising DNA encoding humanimmunoglobulin heavy and kappa light chain variable regions with a humanBCMA antigen.

Following immunization, splenocytes were harvested from each mouse andeither (1) fused with mouse myeloma cells to preserve their viabilityand form hybridoma cells and screened for BCMA specificity, or (2)B-cell sorted (as described in US 2007/0280945A1) using a human BCMAfragment as the sorting reagent that binds and identifies reactiveantibodies (antigen-positive B cells).

Chimeric antibodies to BCMA were initially isolated having a humanvariable region and a mouse constant region. The antibodies werecharacterized and selected for desirable characteristics, includingaffinity, selectivity, etc. If necessary, mouse constant regions werereplaced with a desired human constant region, for example wild-type ormodified IgG1 or IgG4 constant region, to generate a fully humananti-BCMA antibody. While the constant region selected may varyaccording to specific use, high affinity antigen-binding and targetspecificity characteristics reside in the variable region.

Heavy and Light Chain Variable Region Amino Acid and Nucleic AcidSequences of Anti-BCMA Antibodies:

Table 1 sets forth the amino acid sequence identifiers of the heavy andlight chain variable regions and CDRs of selected anti-BCMA antibodiesof the invention. The corresponding nucleic acid sequence identifiersare set forth in Table 2.

TABLE 1 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 mAb16711 2 4 6 8 10 12 1416 mAb16716 18 20 22 24 26 28 30 32 mAb16732 34 36 38 40 42 44 46 48mAb16747 50 52 54 56 58 60 62 64 mAb21581 66 68 70 72 74 76 78 80mAb21587 122 123 mAb21589 124 125

TABLE 2 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 mAb16711 1 3 57 9 11 13 15 mAb16716 17 19 21 23 25 27 29 31 mAb16732 33 35 37 39 41 4345 47 mAb16747 49 51 53 55 57 59 61 63 mAb21581 65 67 69 71 73 75 77 79

Example 2: Generation of Anti-CD3 Antibodies

Anti-CD3 antibodies were generated as described in WO 2017/053856, whichis herein incorporated by reference. Two such anti-CD3 antibodies wereselected from the production of bispecific anti-BCMA x anti-CD3antibodies in accordance with the present invention. Table 3 sets forththe amino acid sequence identifiers of the heavy and light chainvariable regions and CDRs of selected anti-CD3 antibodies. Thecorresponding nucleic acid sequence identifiers are set forth in Table4. Other anti-CD3 antibodies for use in preparing bispecific antibodiesin accordance with the present invention can be found in, e.g., WO2014/047231.

TABLE 3 Amino Acid Sequence Identifiers Antibody SEQ ID NOs: DesignationHCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 mAb7221G 90 92 94 96 82 8486 88 mAb7221G20 98 100 102 104 82 84 86 88

TABLE 4 Nucleic Acid Sequence Identifiers Antibody SEQ ID NOs:Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3 mAb7221G 89 9193 95 81 83 85 87 mAb7221G20 97 99 101 103 81 83 85 87

Example 3: Generation of Bispecific Antibodies that Bind BCMA and CD3

The present invention provides bispecific antigen-binding molecules thatbind CD3 and BCMA; such bispecific antigen-binding molecules are alsoreferred to herein as “anti-BCMA x anti-CD3 or anti-CD3xBCMA oranti-BCMA x anti-CD3 bispecific molecules.” The anti-BCMA portion of theanti-BCMA x anti-CD3 bispecific molecule is useful for targeting tumorcells that express BCMA (also known as CD269), and the anti-CD3 portionof the bispecific molecule is useful for activating T-cells. Thesimultaneous binding of BCMA on a tumor cell and CD3 on a T-cellfacilitates directed killing (cell lysis) of the targeted tumor cell bythe activated T-cell.

Bispecific antibodies comprising an anti-BCMA-specific binding domainand an anti-CD3-specific binding domain were constructed using standardmethodologies, wherein the anti-BCMA antigen binding domain and theanti-CD3 antigen binding domain each comprise different, distinct HCVRspaired with a common LCVR. In exemplified bispecific antibodies, themolecules were constructed utilizing a heavy chain from an anti-CD3antibody, a heavy chain from an anti-BCMA antibody and a common lightchain from the anti-CD3 antibody (10082). In other instances, thebispecific antibodies may be constructed utilizing a heavy chain from ananti-CD3 antibody, a heavy chain from an anti-BCMA antibody and anantibody light chain known to be promiscuous or pair effectively with avariety of heavy chain arms.

TABLE 5 Summary of Component Parts of Anti-BCMA × Anti-CD3 BispecificAntibodies Anti-BCMA Anti-CD3 Antigen-Binding Antigen-Binding BispecificDomain Domain Common Antibody Heavy Chain Heavy Chain Light ChainIdentifier Variable Region Variable Region Variable Region bsAb25441D9mAb21581 mAb7221G mAb7221G (also referred to as REGN5458) bsAb25442DmAb21581 mAb7221G20 mAb7221G20 (also referred to as REGN5459)

Table 6 shows the amino acid sequence identifiers for the bispecificanti-BCMA x anti-CD3 antibodies exemplified herein.

TABLE 6 Amino Acid Sequences of Anti-BCMA × Anti-CD3 BispecificAntibodies Anti-BCMA Anti-CD3 Common First Antigen-Binding SecondAntigen-Binding Light Chain Variable Bispecific Domain Domain RegionAntibody HC HCDR HCDR HCDR HC HCDR HCDR HCDR LC LCDR LCDR LCDRIdentifier VR 1 2 3 VR 1 2 3 VR 1 2 3 bsAb25441D 66 68 70 72 90 92 94 9682 84 86 88 (REGN5458) bsAb25442D 66 68 70 72 98 100 102 104 82 84 86 88(REGN5459)

Example 4: Surface Plasmon Resonance Derived Binding Affinities andKinetic Constants of Anti-BCMA Antibodies and Anti-BCMA x Anti-CD3Bispecific Antibodies

Equilibrium dissociation constants (K_(D) values) for hBCMA.mmh (SEQ IDNO: 106) binding to purified anti-BCMA mAbs and anti-BCMA x anti-CD3bispecific mAbs were determined using a real-time surface plasmonresonance biosensor using a Biacore 4000 instrument. The CM5 Biacoresensor surface was derivatized by amine coupling with a monoclonal mouseanti-human Fc antibody (GE, # BR-1008-39) to capture purified anti-BCMAmAbs and anti-BCMA x anti-CD3 bispecific mAbs. All Biacore bindingstudies were performed in a buffer composed of 0.01M HEPES pH 7.4, 0.15MNaCl, 3 mM EDTA, 0.05% v/v Surfactant P20 (HBS-ET running buffer). Formonomeric affinities, different concentrations of the extracellulardomain of human BCMA expressed with C-terminal myc-myc-hexahistidine tag(human BCMA-MMH; SEQ ID NO: 106) or monkey BCMA expressed withC-terminal myc-myc-hexahistidine tag (monkey BCMA-MMH; SEQ ID NO: 110)were prepared in HBS-ET running buffer (ranging from 90 to 1.11 nM,3-fold dilutions). For dimeric affinities, different concentrations ofthe extracellular domain of human BCMA expressed with C-terminal mFc tag(human BCMA-MFC; SEQ ID NO: 108) monkey BCMA expressed with C-terminalmFc tag (monkey BCMA-MFC; SEQ ID NO: 112) prepared in HBS-ET runningbuffer (ranging from 30 to 0.37 nM, 3-fold dilutions) or 30 nM BCMAexpressed with C-terminal mFc tag (mouse BCMA-MFC; SEQ ID NO: 114) wereprepared. Antigen samples were then injected over the anti-BCMA andanti-BCMA x anti-CD3 bispecific mAbs captured surfaces at a flow rate of30 μL/minute. Antibody-reagent association was monitored for 5 minuteswhile dissociation in HBS-ET running buffer was monitored for 10minutes. All of the binding kinetics experiments were performed at 25°C. Kinetic association (k_(a)) and dissociation (k_(d)) rate constantswere determined by fitting the real-time sensorgrams to a 1:1 bindingmodel using Scrubber 2.0c curve fitting software. Binding dissociationequilibrium constants (K_(D)) and dissociative half-lives (t %) werecalculated from the

${{K_{D}(M)} = \frac{kd}{ka}},{{{and}\mspace{14mu} t\frac{1}{2}} = \frac{\ln (2)}{60*{kd}}}$

As shown in Table 7, at 25° C., all of the anti-BCMA antibodies of theinvention bound to human BCMA-MMH with K_(D) values ranging from 1.06 nMto 3.56 nM. As shown in Table 8, at 25° C., all of the anti-BCMAantibodies of the invention bound to human BCMA-MFC with K_(D) valuesranging from 22.3 pM to 103 pM. As shown in Table 9, at 25° C., two ofthe anti-BCMA antibodies of the invention bound to monkey BCMA-MMH withK_(D) values ranging from 38.8 nM to 49.92 nM. As shown in Table 10, at25° C., four of the anti-BCMA antibodies of the invention bound tomonkey BCMA-MFC with K_(D) values ranging from 148 pM to 14.7 nM. Asshown in Table 11, at 25° C., four of the anti-BCMA antibodies of theinvention bound to mouse BCMA-MFC with K_(D) values ranging from 677 pMto 18.8 nM.

TABLE 7 Binding Kinetics parameters of anti-BCMA monoclonal antibodiesbinding to human BCMA-MMH at 25° C. mAb 90 nM Capture hBCMA.mmh t1/2REGN # Ab PID # (RU) Bind (RU) ka (1/Ms) kd (1/s) KD (M) (min) REGN5458bsAb25441D 437.5 ± 1.1 19.9 8.27E+05 8.74E−04 1.06E−09 13.2 REGN5459bsAb25442D 384.8 ± 1.4 17.0 7.30E+05 1.01E−03 1.38E−09 11.5 mAb16711275.0 ± 2.8 22.2 2.01E+06 3.47E−03 1.73E−09 3.3 mAb16716 310.3 ± 2.226.4 8.41E+05 2.99E−03 3.56E−09 3.9 REGN4514 mAb16732 284.1 ± 0.9 25.31.06E+06 2.85E−03 2.69E−09 4.1 REGN4515 mAb16747 332.5 ± 0.9 31.48.69E+05 2.47E−03 2.84E−09 4.7

TABLE 8 Binding Kinetics parameters of anti-BCMA monoclonal antibodiesbinding to human BCMA-MFC at 25° C. mAb 30 nM Capture hBCMA.mFc t1/2REGN # Ab PID # (RU) Bind (RU) ka (1/Ms) kd (1/s) KD (M) (min) REGN5458bsAb25441D 437.9 ± 0.1 106.8 4.48E+05 ≤1E−5 2.23E−11 ≤1155 REGN5459bsAb25442D 385.2 ± 0.1 96.8 4.49E+05 ≤1E−5 2.23E−11 ≤1155 mAb16711 268.9± 1.4 113.5 1.85E+06 1.90E−04 1.03E−10 60.8 mAb16716 303.4 ± 1.2 120.38.62E+05 8.35E−05 9.68E−11 138.4 REGN4514 mAb16732 282.3 ± 1.0 124.11.07E+06 4.53E−05 4.22E−11 255.2 REGN4515 mAb16747 327.3 ± 1.5 146.01.41E+06 8.95E−05 6.33E−11 129.0

TABLE 9 Binding Kinetics parameters of anti-BCMA monoclonal antibodiesbinding to monkey BCMA-MMH at 25° C. mAb 90 nM Capture mfBCMA.mmh t1/2REGN # Ab PID # (RU) Bind (RU) ka (1/Ms) kd (1/s) KD (M) (min) REGN5458bsAb25441D 438.2 ± 0.9 14.8 1.82E+05 9.09E−03 4.99E−08 1.3 REGN5459bsAb25442D 384.6 ± 1.4 12.7 2.23E+05 8.64E−03 3.88E−08 1.3 mAb16711263.5 ± 1.7 −0.5 NB NB NB NB mAb16716 301.8 ± 0.5 0.8 NB NB NB NBREGN4514 mAb16732 279.1 ± 0.8 1.1 NB NB NB NB REGN4515 mAb16747 326.2 ±0.5 1.9 NB NB NB NB

TABLE 10 Binding Kinetics parameters of anti-BCMA monoclonal antibodiesbinding to monkey BCMA-MFC at 25° C. mAb 30 nM Capture mfBCMA.mFc t1/2REGN # Ab PID # (RU) Bind (RU) ka (1/Ms) kd (1/s) KD (M) (min) REGN5458bsAb25441D 437.9 ± 1.1 107.7 5.28E+05 8.80E−05 1.67E−10 131.2 REGN5459bsAb25442D  386.2 ± 0.22 97.0 4.82E+05 7.15E−05 1.48E−10 161.6 mAb16711259.4 ± 1.4 0.9 NB NB NB NB mAb16716 300.8 ± 0.6 3.2 IC IC IC ICREGN4514 mAb16732 276.9 ± 1.1 40.3 4.92E+05 7.24E−03 1.47E−08 1.6REGN4515 mAb16747 324.4 ± 0.7 101.3 2.13E+06 7.16E−03 3.37E−09 1.6

TABLE 11 Binding Kinetics parameters of anti-BCMA monoclonal antibodiesbinding to mouse BCMA-MFC at 25° C. mAb 30 nM Capture mBCMA.mFc t1/2REGN # Ab PID # (RU) Bind (RU) ka (1/Ms) kd (1/s) KD (M) (min) REGN5458bsAb25441D 438.8 2.7 NB NB NB NB REGN5459 bsAb25442D 383.9 2.4 NB NB NBNB mAb16711 257.0 90.0 1.07E+06 1.10E−03 1.02E−09 10.5 mAb16716 300.033.4 2.05E+05 3.85E−03 1.88E−08 3.0 REGN4514 mAb16732 276.1 109.63.97E+05 2.69E−04 6.77E−10 43.0 REGN4515 mAb16747 323.1 107.6 9.47E+054.18E−03 4.42E−09 2.8

Example 5: FACS Binding of Anti-BCMA x Anti-CD3 Bispecific Antibodies toHuman and Cynomolgous CD3 Expressing Cells

Flow cytometric analysis was utilized to determine binding of BCMAxCD3bispecific antibodies to human and cynomolgus CD3 (Jurkat cells, mfCD3engineered Jurkat cells, primary human CD8+ and cynomolgus CD8+ Tcells). Briefly, 1e05 cells/well were incubated in the presence of FACSwash with block (PBS+1% filtered FBS+5% mouse serum) with a serialdilution of BCMAxCD3 and control antibodies for 30 minutes on ice. Afterincubation, the cells were washed twice with cold FACS wash (PBS+1%filtered FBS) and bound antibody was detected by incubating withAlexa647-conjugated anti-human secondary antibody on ice for anadditional 30 minutes. Wells containing no antibody or secondary onlywere used as a control. For the detection of monkey and human T cells, acocktail of human and cynomolgus cross-reactive antibodies to CD4, CD8and CD16 was added to the anti-human secondary. After incubation, cellswere washed, re-suspended in 200 μL cold PBS containing 1% filtered FBSand analyzed by flow cytometry on a BD FACS Canto II. Cells were gatedby FSC-H by FSC-A to select singlet events, followed by side and forwardscatters to select for live events. For monkey T cells, additionalgating on CD8+/CD16− cells was performed.

EC50 values for FACS binding were calculated using 4-parameternon-linear regression analysis in Prism software.

Jurkat cells are a human CD3 expressing T cell lymphoblastic cell line.REGN5458 bound to human CD3 on Jurkat cells and primary human CD8+ Tcells with median EC50s 1.50×10⁻⁸ M and 3.20×10⁻⁸ M respectively.Binding of REGN5459 was to human CD3 was weaker, with median EC50 of5.58×10⁻⁷ M to Jurkat cells and 4.71×10⁻⁶ to primary human CD8+ T cells.Utilizing CRISPR/Cas9 technology, a Jurkat cell line was engineered toexpress cynomolgus CD3ε and CD3δ chains in place of the human versions.Median EC50 of binding of REGN5458 to the mfCD3 engineered Jurkat cellline was 1.51×10⁻⁸ M and to primary cynomolgus CD8+ T cells was4.66×10⁻⁸ M. REGN5459 did not bind to mfCD3 expressing cells.

No binding was observed on any cell line for the negative isotypecontrol antibody, designated mAb15260.

TABLE 12 Binding to CD3 expressing cells: Median EC50 Human Mf (Cyno)Jurkat- Jurkat- CD8+ CD8+ hCD3 mfCD3 T cells T cells REGN EC50 [M] nEC50 [M] n EC50 [M] n EC50 [M] n REGN5458 1.50E−08 5 1.51E−08 2 3.20E−081 4.66E−08 1 REGN5459 5.58E−07 5 No Binding 2 4.71E−06 1 No binding 1

Example 6: FACS Binding Assay to Assess Cell Surface Antigen BindingCapacity

The ability of the anti-BCMA x CD3 antibody, mAb25442D, to bind thesurface of BCMA positive multiple myeloma (NCI-H929, MM.1S, OPM-2, andRPMI-8226), BCMA positive lymphoma (Raji and Daudi), and BCMA negative(HEK293) cells was determined via flow cytometry. Cells were harvestedfrom the flasks using cell dissociation buffer (Millipore, Cat #S-004-C) and plated in staining buffer (PBS, without Calcium andMagnesium (Irving 9240)+2% FBS (ATCC 30-2020) at a density of 500,000cells per well in a 96 well V-Bottom plate. Cells were stained for 30mins at 4° C. with two-fold serial dilutions of an Alexa647 conjugatedanti-BCMA x CD3 antibody (mAb25442D-A647) or an Alexa 647 conjugatedisotype control with the same CD3 binding arm paired with an irrelevanttumor targeting arm (Isotype-A647). Cells were washed twice withstaining buffer and labeled with the LIVE/DEAD™ Fixable Green Dead CellStain Kit (Invitrogen, L34970) according to manufacture instructions todiscriminate between live and dead cells. Cells were then washed andfixed for 25 mins at 4° C. using a 50% solution of BD Cytofix (BD, Cat#554655) diluted in PBS. Samples were run on the Accuri C6 flowcytometer (BD Biosciences) and analyzed in Flowjo 10.2 (Tree Star).After gating for live cells and single cells, the mean fluorescentintensity (MFI) was determined, and MFI values were plotted in GraphpadPrism using a four-parameter logistic equation over a 10-point responsecurve to calculate EC₅₀s. The zero condition for each dose-responsecurve is also included in the analysis as a continuation of the two-foldserial dilution and is represented as the lowest dose. The signal tonoise (S/N) is determined by taking the ratio of the mAb25442D-A647 MFIto the Isotype-A647 MFI. (Table 13). The mAb25442D-A647 S/N ranged from2 to 470 and the EC₅₀ values ranged from 27 to 83 nM. No detectablebinding was observed on HEK293 cells.

TABLE 13 Binding to Cells mAb25442D-A647 mAb25442D-A647 Cell Line S/NEC₅₀ (nM) NCI-H929 470 79 MM.1S 43 83 OPM-2 19 57 RPMI-8226 9 27 Daudi 3ND Raji 2 ND HEK293 1 ND ND = not determined due to non-sigmoidal curves

Example 7: T-Cell Activation Via Bispecific Anti-BCMA x Anti-CD3Antibodies in the Presence of BCMA-Expressing Cells

Activity of the anti-BCMA x anti-CD3 bispecific antibodies were assessedin a Jurkat/NFATLuc reporter bioassay utilizing several cell lines withvarying levels of BCMA surface expression. The Jurkat cells wereengineered to express an NFAT-luciferase reporter (Jurkat/NFATLuc.3C7),and 50,000 Jurkat reporter cells were combined with 50,000 BCMA positive(Daudi, MM1-S, NCI-H929, OPM-2, RPMI-8226, MOLP-8, or Raji) or BCMAnegative (HEK293) cells in Thermo Nunclon delta 96 well white microwellplates (Thermo Scientific, Cat #136102) in 50 ul of assay media (RPMImedia with 10% FBS and 1% P/S/G). Three-fold serial dilutions of theBCMA x CD3 bispecific antibodies (mAb25441D or mAb25442D), or a bivalentanti-BCMA antibody (mAb21581) were immediately added in 50 uL of assaybuffer. The plates were gently agitated and incubated in a 37° C., 5%CO₂ incubator for 4-6 hours. NFAT-Luciferase activity was determinedusing Promega One-Glo (Cat # E6130) and a Perkin Elmer Envision platereader. RLU were plotted in GraphPad Prism using a four-parameterlogistic equation over a 12-point response curve to calculate EC₅₀values. The no antibody treatment condition for each dose-response curveis also included in the analysis as a continuation of the three-foldserial dilution and is represented as the lowest dose. The signal tonoise (S:N) is determined by taking the ratio of the highest RLU on thecurve to the lowest.

mAb25441D activated Jurkat/NFATLuc cells in the presence of BCMAexpressing cells with EC50s ranging from 0.61 nM to 2.1 nM and S:Nranging from 8 to 123. mAb25442D activated Jurkat/NFATLuc cells in thepresence of BCMA expressing cells with EC50s ranging from 2.6 nM to 11nM and S:N ranging from 7 to 120. The BCMA x CD3 bispec mAb25441D withthe higher affinity CD3 binding arm was consistently more potent thanmAb25442D with a lower affinity CD3 binding arm; whereas, the S:N wassimilar for the two bispecifics. Neither antibody activatedJurkat/NFATLuc cells in the presence of HEK293 cells, and controlbispecific antibodies did not significantly increase Jurkat reporteractivity with any of the tested cell lines. The results are shown inTables 14A and 14B, below.

TABLE 14A Activation of T-Cells Daudi MM1-S NCI-H929 OPM-2 AntibodiesEC50 S:N EC50 S:N EC50 S:N EC50 S:N bsAb25441D 2.1E−9 43 1.2E−9 1656.8E−10 39 6.6E−10 8 bsAb25442D 7.9E−9 25 4.4E−9 120 2.7E−9  32 2.6E−9 7 mAb21581 ND 1 ND 1 ND 1 ND 1

TABLE 14B Activation of T-Cells RPMI-8226 MOLP-8 Raji HEK293 AntibodiesEC50 S:N EC50 S:N EC50 S:N EC50 S:N bsAb25441D 6.1E−10 55 1.4E−9 321.6E−9 123 ND 1 bsAb25442D 2.6E−9  42 1.1E−8 31 7.4E−9 78 ND 1 mAb21581ND 1 ND 1 ND 1 ND 1

Example 8: FACS Based Cytotoxicity Assay to Assess T Cell-MediatedKilling of BCMA-Expressing Multiple Myeloma Cells in the Presence ofAnti-BCMA x Anti-CD3 Bispecific Antibodies

Antibody binding capacity (ABC) of a commercially available anti-humanBCMA antibody (clone 19F2) was determined on a panel of multiple myelomacell lines using a Quantum Simply Cellular anti-human IgG kit andfollowing the manufacturer's instructions (Bangs Laboratories).

Briefly, multiple myeloma (MM) cell lines (H929, MM1S, U266, MOLP8 andRPMI8226) and Quantum Simply Cellular beads were incubated for 30minutes at 4° C. with a titration of APC conjugated anti-hBCMA-19F2antibody. After incubation, cells and beads were washed three times,re-suspended in 200 μL cold PBS containing 1% filtered FBS and analyzedby flow cytometry. Using the QuickCal® template (Bangs Labs), the ABC ofa saturating level of anti-BCMA 19F2 for each cell line was interpolatedfrom the standard curve generated by the channel intensity of the beadpopulations at saturation.

Killing of BCMA expressing target cells by resting human or cynomolgusmonkey T cells was determined by flow cytometry. Briefly, human orcynomolgus monkey peripheral blood mononuclear cells (PBMC) were platedin supplemented RPMI (human) or X-Vivo (cyno) media at 1×10⁸ cells/mLand incubated overnight at 37° C. in order to enrich for lymphocytes bydepleting adherent macrophages, dendritic cells, and some monocytes. Thenext day, BCMA expressing target cells were labeled with 1 uM of VioletCellTrace and co-incubated with adherent cell-depleted PBMC(effector/target cell 4:1 ratio) and a serial dilution of BCMAxCD3bispecifics, or control antibodies at 37° C. After 48-72 hrs, cells wereremoved from cell culture plates, stained with a cocktail phenotypingantibodies and live/dead cell viability dye, and analyzed by FACS. Inorder to quantify the number of live target cells present in the wells,20 μl CountBright absolute counting beads were added to the wells justprior to acquisition. For the assessment of specificity of killing,cells were gated on Violet cell tracker labeled populations. Percentsurvival of target cells was calculated as followed: Targetsurvival=(R₁/R₂)*100, where R₁=absolute number of live target cells inthe presence of effector cells and antibody, and R₂=number of livetarget cells only (cultured without effector cells or test antibody).

Human CD8+ T cells were gated as CD45+/CD14−/CD4−/CD8+. Cynomolgus CD8+T cells were gated as CD45+/CD20−/CD14−/CD4−/CD8+ T cell activation wasreported as the percent of CD25+ or CD69+ T cells out of total CD8+ Tcells.

EC50 values for target cell survival and T cell activation werecalculated using 4-parameter non-linear regression analysis in Prismsoftware.

Anti-BCMA x anti-CD3 bispecific antibodies were tested for their abilityto activate resting human and cynomolgus T cells to kill a panel of BCMAexpressing cells with differing surface BCMA levels. With resting humanT cells as effector cells, REGN5458 mediated killing of 5 different BCMAcell lines with EC₅₀ values ranging from 7.07×10⁻¹⁰ M to 3.45×10⁻¹¹ M.REGN5459 showed killing of the same 5 cell lines with EC50s valuesranging from 1.66×10⁻⁹ M to 1.06×10⁻¹⁰ M. EC₅₀s for T cell activation,as measured by CD25 upregulation on CD8+ T cells were similar to killingEC₅₀s. Modest T cell activation was observed in the presence of 1-armCD3 isotype control mAb17664D, but only for the U266 cell line. Nocytotoxicity was observed for the isotype controls tested.

BCMAxCD3 mediated killing by cynomolgus T cells was tested only on theMM cell line H929. The EC₅₀ for cytotoxicity mediated by REGN5458 andREGN5459 was 2.34×10⁻¹¹ and 6.92×10⁻¹¹ respectively. No cytotoxicity orT cell activation was observed for the isotype control antibody mAb15260with either human or cynomolgus effector cells. The results are shown inTables 15A, 15B and 16, below.

TABLE 15A Median EC₅₀, Human Effector Cells H929 (40000 ABC) MM1S (18000ABC) U266 (13000 ABC) REGN# n % Survival % T activation n % Survival % Tactivation n % Survival % T activation REGN5458 3 1.03E−10 2.11E−10 26.46E−11 7.06E−11 1 3.28E−10 1.07E−10 REGN5459 4 3.01E−10 3.00E−10 22.88E−10 4.58E−10 1 1.66E−09 4.69E−10

TABLE 15B Median EC₅₀, Human Effector Cells RPMI8226 (10000 ABC) Molp8(2000 ABC) REGN# n % Survival % T activation n % Survival % T activationREGN5458 1 3.45E−11 6.49E−11 2 7.07E−10 1.10E−9 REGN5459 1 1.06E−107.50E−10 3 1.36E−09 6.47E−9

TABLE 16 Median EC₅₀, Cynomolgus effector cells H929 REGN# n % Survival% T activation REGN5458 4 2.34E−11 6.83E−11 REGN5459 4 6.92E−11 1.58E−10

Example 9: FACS Cytotoxicity Assay to Autologous T Cell-Mediated Killingof Primary Multiple Myeloma Blast Cells in the Presence of Anti-BCMA xAnti-CD3 Bispecific Antibodies

In order to monitor the specific killing of multiple myeloma cells byflow cytometry, bone marrow mononuclear cells (BMMC) from multiplemyeloma patients were plated on human stromal cells (HS5) and restedovernight at 37 C. Separately, matching patient peripheral bloodmononuclear cells (PBMC) were thawed and cultured in supplemented RPMImedia at 1×10⁶ cells/mL overnight at 37° C. in order to enrich forlymphocytes by depleting adherent cells. The next day, BMMC wereco-incubated with adherent cell-depleted naive PBMC on stromal cells(HS5) and a serial 10× dilution of BCMAxCD3 bispecific or 1-arm CD3isotype control (starting concentration 66.7 nM) at 37° C. Cells wereremoved from cell culture plates at day 3, 4 or 7 and analyzed by FACS.For the assessment of specificity of killing, multiple myeloma cellswere gated as single, live, CD90 negative (to exclude stromal cells),CD2 negative, CD56 positive. CD45 was low on multiple myeloma cells inmost samples except MM455. Percent of live target cells was reported forthe calculation of adjusted survival as follows: Adjustedsurvival=(R1/R2)*100, where R1=% live target cells in the presence ofantibody, and R2=% live target cells in the absence of test antibody.

T cells were gated as CD2 positive, CD56 negative and either CD4 or CD8positive. T cell activation was reported as the percent of CD25+CD4 orCD8 T cells out of total CD4 or CD8 T cells.

BCMAxCD3 bispecific antibodies were tested for their ability to redirectkilling of primary multiple myeloma blast cells by autologous donorPBMC. Maximal BCMAxCD3 mediated cytotoxicity of primary MM blast rangedfrom 52-96%, with EC50s ranging from 9.89×10⁻¹¹ M to 3.67×10⁻⁹ M forREGN5458 and 4.96×10⁻¹⁰ M to 7.94×10⁻⁸ M for REGN5459. T cell activationwas measured by assessing the upregulation of CD25 on CD8+ T cells.EC50s of T cell activation ranged from 3.23×10⁻⁹ to 1.69×10⁻¹⁰. Modestcytotoxicity and T cell activation was observed for the 1-arm CD3 (notarget binding) isotype control. Results are shown in Tables 17A and17B, below.

TABLE 17A MM % lysis % MM % MM % MM lysis at lysis at lysis at SampleDisease E:T length of 66 nM 66 nM 66 nM ID Stage ratio treatmentREGN5458 REGN5459 Isotype MM2 newly diagnosed 1.4 7 days 88 85 27.5MM369 newly diagnosed 0.3 3 days 96 94 0 MM453 newly diagnosed 2.4 3days 82 80 40 MM455 progression, treated 0.4 3 days 63 52 24

TABLE 17B MM lysis EC50 and T cell activation CD25 CD25 MM Lysis MMlysis upreg upreg Sample Disease E:T length of EC50 EC50 EC50 EC50 IDStage ratio treatment REGN5458 REGN5459 REGN5456 REGN5456 MM2 newly 1.47 days 7.47E−10 7.24E−09 Not done Not done diagnosed MM369 newly 0.3 3days 1.07E−10 4.96E−10 1.69E−10  2.03E−10 diagnosed MM453 newly 2.4 3days 9.89E−11 1.19E−09 1.71E−10 3.23E−9 diagnosed MM455 progression, 0.43 days 3.67E−09 7.94E−08 2.06E−10 1.16E−9 treated

Example 10: Anti-BCMA x Anti-CD3 Bispecific Antibodies Prevent Growth ofBCMA-Expressing Tumors (NCI-H929) In Vivo in a Xenogenic Tumor Model

To determine the in vivo efficacy of BCMAxCD3 bispecific antibodies(Abs), a xenogenic tumor study was performed. ImmunodeficientNOD.Cg-Prkdc^(scidSd)II2rgm^(tm1Wji)/SzJ (NSG) mice were subcutaneouslyimplanted with a mixture of 10×10⁶ BCMA-expressing NCI-H929 multiplemyeloma cells and 0.5×10⁶ human peripheral blood mononuclear cells(PBMC) isolated from a normal donor. The mice (n=7 per group) wereimmediately administered a PBS vehicle control, an irrelevant anti-FelD1bivalent isotype control Ab (REGN2759), a CD3-binding control bispecificAb (mAb17664D), a BCMAxCD3 (G; REGN5458) bispecific Ab, or a BCMAxCD3(G20; REGN5459) bispecific Ab at a dose of 4 mg/kg. The mice wereadministered Abs twice per week for a total of three weeks, and tumorgrowth was assessed over 40 days. While BCMA⁺ tumors grew similarly inthe vehicle-, isotype control-, and CD3-binding control-treated mice,both BCMAxCD3 Abs that were tested prevented the growth of tumors invivo.

Implantation and Measurement of Syngeneic Tumors:

NSG mice were subcutaneously implanted with a mixture of 10×10⁶BCMA-expressing NCI-H929 multiple myeloma cells and 0.5×10⁶ PBMC derivedfrom a normal donor. The mice (n=7 per group) were immediatelyadministered a PBS vehicle control, an irrelevant anti-FelD1 bivalentisotype control Ab (REGN2759), a CD3-binding control bispecific Ab(mAb17664D), a BCMAxCD3 (G; REGN5458) bispecific Ab, or a BCMAxCD3 (G20;REGN5459) bispecific Ab at a dose of 4 mg/kg. The mice were administeredAbs twice per week for a total of three weeks. Tumor growth was measuredwith calipers twice per week for the duration of the experiment. Micewere sacrificed 40 days after tumor implantation.

Calculation of Syngeneic Tumor Growth and Inhibition:

In order to determine tumor volume by external caliper, the greatestlongitudinal diameter (length in mm) and the greatest transversediameter (width in mm) were determined. Tumor volumes based on calipermeasurements were calculated by the formula: Volume(mm³)=(length×width²)/2.

BCMAxCD3 bispecific Abs prevented the growth of BCMA⁺ NCI-H929 tumors invivo in a xenogenic tumor model. Results are shown in Table 18, below.

TABLE 18 Average Tumor Size at Various Time Points Antibody (4 mg/kg)Average Tumor Size (mm3) ± SEM on Day 4 PBS (Vehicle Control) 67.1 ± 5.9REGN2759 (Isotype Control) 62.6 ± 3.7 mAb17664D (CD3 Binding Control)76.1 ± 7.6 REGN5458 (BCMA × CD3-G) 39.5 ± 9.1 REGN5459 (BCMA × CD3-G20)26.5 ± 6.2 Average Tumor Size (mm3) ± SEM on Day 7 PBS (Vehicle Control)123.0 ± 25.2 REGN2759 (Isotype Control) 109.7 ± 20.3 mAb17664D (CD3Binding Control) 182.0 ± 19.4 REGN5458 (BCMA × CD3-G)  0 ± 0 REGN5459(BCMA × CD3-G20)  0 ± 0 Average Tumor Size (mm3) ± SEM on Day 11 PBS(Vehicle Control) 361.5 ± 35.7 REGN2759 (Isotype Control) 415.3 ± 11.4mAb17664D (CD3 Binding Control) 449.6 ± 46.6 REGN5458 (BCMA × CD3-G)  0± 0 REGN5459 (BCMA × CD3-G20)  0 ± 0 Average Tumor Size (mm3) ± SEM onDay 14 PBS (Vehicle Control) 581.4 ± 57.9 REGN2759 (Isotype Control)734.3 ± 41.8 mAb17664D (CD3 Binding Control) 741.2 ± 56.0 REGN5458 (BCMA× CD3-G)  0 ± 0 REGN5459 (BCMA × CD3-G20)  0 ± 0 Average Tumor Size(mm3) ± SEM on Day 18 PBS (Vehicle Control) 1033.4 ± 143.7 REGN2759(Isotype Control) 1586.1 ± 101.4 mAb17664D (CD3 Binding Control) 1511.4± 80.7  REGN5458 (BCMA × CD3-G)  0 ± 0 REGN5459 (BCMA × CD3-G20)  0 ± 0Average Tumor Size (mm3) ± SEM on Day 21 PBS (Vehicle Control) 1730.9 ±244.8 REGN2759 (Isotype Control) 2554.7 ± 148.8 mAb17664D (CD3 BindingControl) 2474.0 ± 132.6 REGN5458 (BCMA × CD3-G)  0 ± 0 REGN5459 (BCMA ×CD3-G20)  0 ± 0 Average Tumor Size (mm3) ± SEM on Day 28 PBS (VehicleControl) Euthanized - Not measured REGN2759 (Isotype Control)Euthanized - Not measured mAb17664D (CD3 Binding Control) Euthanized -Not measured REGN5458 (BCMA × CD3-G)  0 ± 0 REGN5459 (BCMA × CD3-G20)  0± 0 Average Tumor Size (mm3) ± SEM on Day 40 PBS (Vehicle Control)Euthanized - Not measured REGN2759 (Isotype Control) Euthanized - Notmeasured mAb17664D (CD3 Binding Control) Euthanized - Not measuredREGN5458 (BCMA × CD3-G)  0 ± 0 REGN5459 (BCMA × CD3-G20)  0 ± 0

Example 11: Anti-BCMA x Anti-CD3 Bispecific Antibodies Prevent Growth ofBCMA-Expressing Tumors (NCI-H929) in a Dose-Dependent Manner in aXenogenic In Vivo Tumor Model

To determine the in vivo efficacy of anti-BCMA x anti-CD3 bispecificantibodies (Abs), a xenogenic tumor study was performed. ImmunodeficientNOD.Cg-Prkc^(scid)II2rg^(tm1Wji)/SzJ (NSG) mice were subcutaneouslyimplanted with a mixture of 10×10⁶ BCMA-expressing NCI-H929 humanmultiple myeloma tumor cells and 0.5×10⁶ human peripheral bloodmononuclear cells (PBMC) isolated from a normal, healthy donor. The mice(n=7 per group) were then immediately administered a PBS vehiclecontrol, a CD3-binding control bispecific Ab (G; mAb17664D) at a dose of4 mg/kg, a CD3-binding control bispecific Ab (G20; REGN4460) at a doseof 4 mg/kg, a BCMAxCD3 (G; REGN5458) bispecific Ab at doses of either 4mg/kg, 0.4 mg/kg, or 0.04 mg/kg, or a BCMAxCD3 (G20; REGN5459)bispecific Ab at doses of either 4 mg/kg, 0.4 mg/kg, or 0.04 mg/kg. Themice were administered these Abs twice per week for a total of sevendoses, and tumor growth was assessed over 60 days. While BCMA⁺ NCI-H929tumors grew similarly in the vehicle- and CD3-binding control-treatedmice, both anti-BCMA x anti-CD3 Abs that were tested prevented thegrowth of tumors in a dose-dependent manner in vivo.

Implantation and Measurement of Xenogenic Tumors:

NSG mice were subcutaneously implanted with a mixture of 10×10⁶BCMA-expressing NCI-H929 multiple myeloma cells and 0.5×10⁶ PBMC derivedfrom a normal, healthy donor. The mice (n=7 per group) were immediatelyadministered a PBS vehicle control, a CD3-binding control bispecific Ab(G; mAb17664D), a CD3-binding control bispecific Ab (G20; REGN4460), aBCMAxCD3 (G; REGN5458) bispecific Ab, or a BCMAxCD3 (G20; REGN5459)bispecific Ab. mAb17664D and REGN4460 were dosed at 4 mg/kg, whileREGN5458 and REGN5459 were administered at either 4 mg/kg, 0.4 mg/kg, or0.04 mg/kg. The mice were administered Abs twice per week for a total ofseven doses. Tumor growth was measured with calipers twice per week forthe duration of the experiment.

Calculation of Xenogenic Tumor Growth and Inhibition:

In order to determine tumor volume by external caliper, the greatestlongitudinal diameter (length in mm) and the greatest transversediameter (width in mm) were determined. Tumor volumes based on calipermeasurements were calculated by the formula: Volume(mm³)=(length×width²)/2.

BCMAxCD3 bispecific Abs prevented the growth of BCMA⁺ NCI-H929 tumors ina dose-dependent manner in this xenogenic in vivo tumor model. Resultsare shown in Table 19, below, and illustrated in FIGS. 1 and 2.

TABLE 19 Average Tumor Size at Various Time Points Antibody TreatmentAverage Tumor Size (mm3) ± SEM on Day 4 PBS (Vehicle Control) 60.1 ±7.9  mAb17664D (CD3 Binding Control-G) - 4 mg/kg 42.5 ± 4.7  REGN4460(CD3 Binding Control-G20) - 4 mg/kg 52.0 ± 5.9  REGN5458 (BCMA ×CD3-G) - 4 mg/kg 18.0 ± 1.2  REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 31.9 ±2.0  REGN5458 (BCMA × CD3-G) - 0.04 mg/kg 32.0 ± 2.9  REGN5459 (BCMA ×CD3-G20) - 4 mg/kg 21.8 ± 3.4  REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg19.6 ± 4.4  REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg 33.0 ± 4.4  AverageTumor Size (mm3) ± SEM on Day 7 PBS (Vehicle Control) 138.2 ± 25.1 mAb17664D (CD3 Binding Control-G) - 4 mg/kg 108.6 ± 17.8  REGN4460 (CD3Binding Control-G20) - 4 mg/kg 132.4 ± 21.1  REGN5458 (BCMA × CD3-G) - 4mg/kg 1.3 ± 1.3 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 11.3 ± 3.0  REGN5458(BCMA × CD3-G) - 0.04 mg/kg 30.8 ± 5.5  REGN5459 (BCMA × CD3-G20) - 4mg/kg 8.0 ± 4.3 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 7.3 ± 3.6 REGN5459(BCMA × CD3-G20) - 0.04 mg/kg 8.4 ± 4.0 Average Tumor Size (mm3) ± SEMon Day 12 PBS (Vehicle Control) 545.4 ± 88.7  mAb17664D (CD3 BindingControl-G) - 4 mg/kg 493.4 ± 67.5  REGN4460 (CD3 Binding Control-G20) -4 mg/kg 616.2 ± 84.4  REGN5458 (BCMA × CD3-G) - 4 mg/kg 0 ± 0 REGN5458(BCMA × CD3-G) - 0.4 mg/kg 1.6 ± 1.6 REGN5458 (BCMA × CD3-G) - 0.04mg/kg 71.5 ± 22.4 REGN5459 (BCMA × CD3-G20) - 4 mg/kg 1.7 ± 1.7 REGN5459(BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.04mg/kg 0 ± 0 Average Tumor Size (mm3) ± SEM on Day 15 PBS (VehicleControl) 921.4 ± 147.5 mAb17664D (CD3 Binding Control-G) - 4 mg/kg 874.8± 86.6  REGN4460 (CD3 Binding Control-G20) - 4 mg/kg 1190.7 ± 91.2 REGN5458 (BCMA × CD3-G) - 4 mg/kg 0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4mg/kg 0 ± 0 REGN5458 (BCMA × CD3-G) - 0.04 mg/kg 133.4 ± 50.9  REGN5459(BCMA × CD3-G20) - 4 mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0± 0 REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg 7.9 ± 7.9 Average Tumor Size(mm3) ± SEM on Day 19 PBS (Vehicle Control) 1785.3 ± 282.2  mAb17664D(CD3 Binding Control-G) - 4 mg/kg 1833.4 ± 186.6  REGN4460 (CD3 BindingControl-G20) - 4 mg/kg 2336.5 ± 188.3  REGN5458 (BCMA × CD3-G) - 4 mg/kg0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 0 ± 0 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg 413.7 ± 162.7 REGN5459 (BCMA × CD3-G20) - 4 mg/kg 0± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg 23.1 ± 23.1 Average Tumor Size (mm3) ± SEM on Day22 PBS (Vehicle Control) 2601.5 ± 414.5  mAb17664D (CD3 BindingControl-G) - 4 mg/kg 2878.5 ± 257.6  REGN4460 (CD3 BindingControl-G20) - 4 mg/kg 3374.3 ± 267.2  REGN5458 (BCMA × CD3-G) - 4 mg/kg0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 0 ± 0 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg 669.4 ± 248.5 REGN5459 (BCMA × CD3-G20) - 4 mg/kg 0± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg 69.5 ± 69.5 Average Tumor Size (mm3) ± SEM on Day26 PBS (Vehicle Control) Animals Euthanized mAb17664D (CD3 BindingControl-G) - 4 mg/kg Animals Euthanized REGN4460 (CD3 BindingControl-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA × CD3-G) - 4mg/kg 0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 0 ± 0 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg 1167.0 ± 431.7  REGN5459 (BCMA × CD3-G20) - 4 mg/kg0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg 156.7 ± 156.7 Average Tumor Size (mm3) ± SEM onDay 29 PBS (Vehicle Control) Animals Euthanized mAb17664D (CD3 BindingControl-G) - 4 mg/kg Animals Euthanized REGN4460 (CD3 BindingControl-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA × CD3-G) - 4mg/kg 0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 0 ± 0 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg 1781.8 ± 620.7  REGN5459 (BCMA × CD3-G20) - 4 mg/kg0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg 226.6 ± 226.6 Average Tumor Size (mm3) ± SEM onDay 34 PBS (Vehicle Control) Animals Euthanized mAb17664D (CD3 BindingControl-G) - 4 mg/kg Animals Euthanized REGN4460 (CD3 BindingControl-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA × CD3-G) - 4mg/kg 0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 0 ± 0 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg Animals Euthanized REGN5459 (BCMA × CD3-G20) - 4mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg Animals Euthanized Average Tumor Size (mm3) ± SEMon Day 39 PBS (Vehicle Control) Animals Euthanized mAb17664D (CD3Binding Control-G) - 4 mg/kg Animals Euthanized REGN4460 (CD3 BindingControl-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA × CD3-G) - 4mg/kg 0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 0 ± 0 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg Animals Euthanized REGN5459 (BCMA × CD3-G20) - 4mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg Animals Euthanized Average Tumor Size (mm3) ± SEMon Day 42 PBS (Vehicle Control) Animals Euthanized mAb17664D (CD3Binding Control-G) - 4 mg/kg Animals Euthanized REGN4460 (CD3 BindingControl-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA × CD3-G) - 4mg/kg 0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 0 ± 0 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg Animals Euthanized REGN5459 (BCMA × CD3-G20) - 4mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg Animals Euthanized Average Tumor Size (mm3) ± SEMon Day 46 PBS (Vehicle Control) Animals Euthanized mAb17664D (CD3Binding Control-G) - 4 mg/kg Animals Euthanized REGN4460 (CD3 BindingControl-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA × CD3-G) - 4mg/kg 0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 0 ± 0 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg Animals Euthanized REGN5459 (BCMA × CD3-G20) - 4mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg Animals Euthanized Average Tumor Size (mm3) ± SEMon Day 55 PBS (Vehicle Control) Animals Euthanized mAb17664D (CD3Binding Control-G) -4 mg/kg Animals Euthanized REGN4460 (CD3 BindingControl-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA × CD3-G) - 4mg/kg 0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 0 ± 0 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg Animals Euthanized REGN5459 (BCMA × CD3-G20) - 4mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg Animals Euthanized Average Tumor Size (mm3) ± SEMon Day 60 PBS (Vehicle Control) Animals Euthanized mAb17664D (CD3Binding Control-G) - 4 mg/kg Animals Euthanized REGN4460 (CD3 BindingControl-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA × CD3-G) - 4mg/kg 0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 0 ± 0 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg Animals Euthanized REGN5459 (BCMA × CD3-G20) - 4mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg Animals Euthanized

Example 12: Anti-BCMA x Anti-CD3 Bispecific Antibodies Reduce the Sizeof and Prevent Growth of Established BCMA-Expressing Tumors (NCI-H929)in a Dose-Dependent Manner in a Xenogenic In Vivo Tumor Model

To determine the in vivo efficacy of anti-BCMA x anti-CD3 bispecificantibodies (Abs), a xenogenic tumor study was performed. ImmunodeficientNOD.Cg-Prkdc^(scid)II2rg^(tm1Wji)/SzJ (NSG) mice were subcutaneouslyimplanted with a mixture of 10×10⁶ BCMA-expressing NCI-H929 humanmultiple myeloma tumor cells and 0.5×10⁶ human peripheral bloodmononuclear cells (PBMC) isolated from a normal, healthy donor. Thetumors were allowed to grow and establish for 5 days until they wereapproximately 70 mm³ in size. On day 5, the mice (n=7-8 per group) werethen administered a PBS vehicle control, a CD3-binding controlbispecific Ab (G; mAb17664D) at a dose of 4 mg/kg, a CD3-binding controlbispecific Ab (G20; REGN4460) at a dose of 4 mg/kg, a BCMAxCD3 (G;REGN5458) bispecific Ab at doses of either 4 mg/kg, 0.4 mg/kg, or 0.04mg/kg, or a BCMAxCD3 (G20; REGN5459) bispecific Ab at doses of either 4mg/kg, 0.4 mg/kg, or 0.04 mg/kg. The mice were administered these Abstwice per week for a total of seven doses, and tumor growth was assessedover 55 days. While BCMA⁺ NCI-H929 tumors grew similarly in the vehicle-and CD3-binding control-treated mice, both BCMAxCD3 Abs that were testedshrank established tumors and prevented the growth of tumors in adose-dependent manner in vivo.

Implantation and Measurement of Xenogenic Tumors:

NSG mice were subcutaneously implanted with a mixture of 10×10⁶BCMA-expressing NCI-H929 multiple myeloma cells and 0.5×10⁶ PBMC derivedfrom a normal, healthy donor. The tumors were allowed to grow andestablish for 5 days until they were approximately 70 mm³ in size. Onday 5, the mice (n=7-8 per group) were then administered a PBS vehiclecontrol, a CD3-binding control bispecific Ab (G; mAb17664D), aCD3-binding control bispecific Ab (G20; REGN4460), a BCMAxCD3 (G;REGN5458) bispecific Ab, or a BCMAxCD3 (G20; REGN5459) bispecific Ab.mAb17664D and REGN4460 were dosed at 4 mg/kg, while REGN5458 andREGN5459 were administered at either 4 mg/kg, 0.4 mg/kg, or 0.04 mg/kg.The mice were administered Abs twice per week for a total of sevendoses. Tumor growth was measured with calipers twice per week for theduration of the experiment.

Calculation of Xenogenic Tumor Growth and Inhibition:

In order to determine tumor volume by external caliper, the greatestlongitudinal diameter (length in mm) and the greatest transversediameter (width in mm) were determined. Tumor volumes based on calipermeasurements were calculated by the formula: Volume(mm³)=(length×width²)/2.

Anti-BCMA x anti-CD3 bispecific antibodies reduced the size of andprevented the growth of established BCMA⁺ NCI-H929 tumors in adose-dependent manner in this xenogenic in vivo tumor model. Results areshown in Table 20, below, and illustrated in FIGS. 3 and 4.

TABLE 20 Average Tumor Size at Various Time Points Antibody TreatmentAverage Tumor Size (mm3) ± SEM on Day 5 PBS (Vehicle Control) 61.5 ± 6.4mAb17664D (CD3 Binding Control-G) - 4 mg/kg 63.7 ± 5.4 REGN4460 (CD3Binding Control-G20) - 4 mg/kg 62.6 ± 3.6 REGN5458 (BCMA × CD3-G) - 4mg/kg  71.9 ± 10.3 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 69.3 ± 7.3REGN5458 (BCMA × CD3-G) - 0.04 mg/kg 58.1 ± 5.6 REGN5459 (BCMA ×CD3-G20) - 4 mg/kg 61.8 ± 5.2 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 69.5± 4.1 REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg 74.9 ± 6.4 Average TumorSize (mm3) ± SEM on Day 8 PBS (Vehicle Control) 124.3 ± 17.3 mAb17664D(CD3 Binding Control-G) - 4 mg/kg 145.3 ± 22.0 REGN4460 (CD3 BindingControl-G20) - 4 mg/kg 170.7 ± 15.5 REGN5458 (BCMA × CD3-G) - 4 mg/kg 64.7 ± 16.4 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 120.3 ± 16.3 REGN5458(BCMA × CD3-G) - 0.04 mg/kg 130.3 ± 16.7 REGN5459 (BCMA × CD3-G20) - 4mg/kg 45.8 ± 9.8 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 171.9 ± 23.2REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg 152.3 ± 20.0 Average Tumor Size(mm3) ± SEM on Day 12 PBS (Vehicle Control) 565.7 ± 64.7 mAb17664D (CD3Binding Control-G) - 4 mg/kg 585.0 ± 64.4 REGN4460 (CD3 BindingControl-G20) - 4 mg/kg 706.8 ± 46.3 REGN5458 (BCMA × CD3-G) - 4 mg/kg 19.5 ± 10.9 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 262.7 ± 61.6 REGN5458(BCMA × CD3-G) - 0.04 mg/kg 525.9 ± 71.5 REGN5459 (BCMA × CD3-G20) - 4mg/kg 11.5 ± 8.9 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 233.8 ± 63.5REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg 462.5 ± 57.7 Average Tumor Size(mm3) ± SEM on Day 15 PBS (Vehicle Control) 1150.4 ± 105.7 mAb17664D(CD3 Binding Control-G) - 4 mg/kg 1041.4 ± 101.3 REGN4460 (CD3 BindingControl-G20) - 4 mg/kg 1298.4 ± 71.0  REGN5458 (BCMA × CD3-G) - 4 mg/kg 25.6 ± 19.2 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg  476.2 ± 133.5 REGN5458(BCMA × CD3-G) - 0.04 mg/kg 1031.2 ± 164.3 REGN5459 (BCMA × CD3-G20) - 4mg/kg  0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg  327.2 ± 135.6REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg 1094.2 ± 78.9  Average Tumor Size(mm3) ± SEM on Day 19 PBS (Vehicle Control) 2621.3 ± 190.9 mAb17664D(CD3 Binding Control-G) - 4 mg/kg 2557.5 ± 241.1 REGN4460 (CD3 BindingControl-G20) - 4 mg/kg 3383.3 ± 183.1 REGN5458 (BCMA × CD3-G) - 4 mg/kg 40.6 ± 32.8 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 1347.5 ± 334.7 REGN5458(BCMA × CD3-G) - 0.04 mg/kg 2467.5 ± 370.0 REGN5459 (BCMA × CD3-G20) - 4mg/kg  0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg  606.2 ± 288.8REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg 2412.5 ± 184.6 Average Tumor Size(mm3) ± SEM on Day 22 PBS (Vehicle Control) 3717.9 ± 214.5 mAb17664D(CD3 Binding Control-G) - 4 mg/kg 3688.9 ± 272.0 REGN4460 (CD3 BindingControl-G20) - 4 mg/kg 4492.2 ± 344.0 REGN5458 (BCMA × CD3-G) - 4 mg/kg 78.3 ± 60.8 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 2068.5 ± 465.0 REGN5458(BCMA × CD3-G) - 0.04 mg/kg 3745.7 ± 541.2 REGN5459 (BCMA × CD3-G20) - 4mg/kg  0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg  815.4 ± 387.1REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg 3285.9 ± 227.3 Average Tumor Size(mm3) ± SEM on Day 27 PBS (Vehicle Control) Animals Euthanized mAb17664D(CD3 Binding Control-G) - 4 mg/kg Animals Euthanized REGN4460 (CD3Binding Control-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA ×CD3-G) - 4 mg/kg  252.3 ± 185.1 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 3463.9 ± 1025.0 REGN5458 (BCMA × CD3-G) - 0.04 mg/kg 1589.1 ± 0   REGN5459 (BCMA × CD3-G20) - 4 mg/kg  0 ± 0 REGN5459 (BCMA × CD3-G20) -0.4 mg/kg 1849.9 ± 903.1 REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg AnimalsEuthanized Average Tumor Size (mm3) ± SEM on Day 30 PBS (VehicleControl) Animals Euthanized mAb17664D (CD3 Binding Control-G) - 4 mg/kgAnimals Euthanized REGN4460 (CD3 Binding Control-G20) - 4 mg/kg AnimalsEuthanized REGN5458 (BCMA × CD3-G) - 4 mg/kg  411.3 ± 307.2 REGN5458(BCMA × CD3-G) - 0.4 mg/kg  2144.2 ± 2144.2 REGN5458 (BCMA × CD3-G) -0.04 mg/kg 2886.5 ± 0    REGN5459 (BCMA × CD3-G20) - 4 mg/kg  0 ± 0REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg  661.8 ± 490.1 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg Animals Euthanized Average Tumor Size (mm3) ± SEMon Day 35 PBS (Vehicle Control) Animals Euthanized mAb17664D (CD3Binding Control-G) - 4 mg/kg Animals Euthanized REGN4460 (CD3 BindingControl-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA × CD3-G) - 4mg/kg  633.5 ± 473.5 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg  0 ± 0 REGN5458(BCMA × CD3-G) - 0.04 mg/kg Animals Euthanized REGN5459 (BCMA ×CD3-G20) - 4 mg/kg  0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg  996.8 ±771.0 REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg Animals Euthanized AverageTumor Size (mm3) ± SEM on Day 40 PBS (Vehicle Control) AnimalsEuthanized mAb17664D (CD3 Binding Control-G) - 4 mg/kg AnimalsEuthanized REGN4460 (CD3 Binding Control-G20) - 4 mg/kg AnimalsEuthanized REGN5458 (BCMA × CD3-G) - 4 mg/kg  369.5 ± 369.5 REGN5458(BCMA × CD3-G) - 0.4 mg/kg  0 ± 0 REGN5458 (BCMA × CD3-G) - 0.04 mg/kgAnimals Euthanized REGN5459 (BCMA × CD3-G20) - 4 mg/kg  0 ± 0 REGN5459(BCMA × CD3-G20) - 0.4 mg/kg  375.6 ± 375.6 REGN5459 (BCMA × CD3-G20) -0.04 mg/kg Animals Euthanized Average Tumor Size (mm3) ± SEM on Day 55PBS (Vehicle Control) Animals Euthanized mAb17664D (CD3 BindingControl-G) - 4 mg/kg Animals Euthanized REGN4460 (CD3 BindingControl-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA × CD3-G) - 4mg/kg  0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg  0 ± 0 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg Animals Euthanized REGN5459 (BCMA × CD3-G20) - 4mg/kg  0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg  0 ± 0 REGN5459 (BCMA× CD3-G20) - 0.04 mg/kg Animals Euthanized

Example 13: Anti-BCMA x Anti-CD3 Bispecific Antibodies Prevent Growth ofBCMA-Expressing Tumors (MOLP-8) in a Dose-Dependent Manner in aXenogenic In Vivo Tumor Model

To determine the in vivo efficacy of anti-BCMA x anti-CD3 bispecificantibodies (Abs), a xenogenic tumor study was performed. ImmunodeficientNOD.Cg-Prkdc^(scid)II2rg^(tm1Wji)/SzJ (NSG) mice were subcutaneouslyimplanted with a mixture of 5×10⁶ BCMA-expressing MOLP-8 human multiplemyeloma tumor cells and 1×10⁶ human peripheral blood mononuclear cells(PBMC) isolated from a normal, healthy donor. The mice (n=7 per group)were then immediately administered a PBS vehicle control, a CD3-bindingcontrol bispecific Ab (G; mAb17664D) at a dose of 4 mg/kg, a CD3-bindingcontrol bispecific Ab (G20; REGN4460) at a dose of 4 mg/kg, a BCMAxCD3(G; REGN5458) bispecific Ab at doses of either 4 mg/kg, 0.4 mg/kg, or0.04 mg/kg, or a BCMAxCD3 (G20; REGN5459) bispecific Ab at doses ofeither 4 mg/kg, 0.4 mg/kg, or 0.04 mg/kg. The mice were administeredthese Abs twice per week for a total of seven doses, and tumor growthwas assessed over 56 days. While the BCMA⁺ MOLP-8 tumors grew similarlyin the vehicle- and CD3-binding control-treated mice, both BCMAxCD3 Absthat were tested prevented the growth of tumors in a dose-dependentmanner in vivo.

Implantation and Measurement of Xenogenic Tumors:

NSG mice were subcutaneously implanted with a mixture of 5×10⁶BCMA-expressing MOLP-8 multiple myeloma cells and 1×10⁶ PBMC derivedfrom a normal, healthy donor. The mice (n=7 per group) were immediatelyadministered a PBS vehicle control, a CD3-binding control bispecific Ab(G; mAb17664D), a CD3-binding control bispecific Ab (G20; REGN4460), aBCMAxCD3 (G; REGN5458) bispecific Ab, or a BCMAxCD3 (G20; REGN5459)bispecific Ab. mAb17664D and REGN4460 were dosed at 4 mg/kg, whileREGN5458 and REGN5459 were administered at either 4 mg/kg, 0.4 mg/kg, or0.04 mg/kg. The mice were administered Abs twice per week for a total ofseven doses. Tumor growth was measured by caliper twice per week for theduration of the experiment

Calculation of Xenogenic Tumor Growth and Inhibition:

In order to determine tumor volume by external caliper, the greatestlongitudinal diameter (length in mm) and the greatest transversediameter (width in mm) were determined. Tumor volumes based on calipermeasurements were calculated by the formula: Volume(mm³)=(length×width²)/2.

Anti-BCMA x anti-CD3 bispecific antibodies prevented the growth of BCMA⁺MOLP-8 tumors in a dose-dependent manner in this xenogenic in vivo tumormodel. Results are shown in Table 21, below, and illustrated in FIGS. 5and 6.

TABLE 21 Average Tumor Size at Various Time Points Antibody TreatmentAverage Tumor Size (mm3) ± SEM on Day 3 PBS (Vehicle Control) 10.3 ±3.0  mAb17664D (CD3 Binding Control-G) - 4 mg/kg 11.6 ± 2.0  REGN4460(CD3 Binding Control-G20) - 4 mg/kg 14.1 ± 3.9  REGN5458 (BCMA ×CD3-G) - 4 mg/kg 12.5 ± 1.3  REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 13.5 ±1.5  REGN5458 (BCMA × CD3-G) - 0.04 mg/kg 9.3 ± 2.4 REGN5459 (BCMA ×CD3-G20) - 4 mg/kg 12.9 ± 1.3  REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg14.0 ± 1.6  REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg 11.7 ± 2.1  AverageTumor Size (mm3) ± SEM on Day 7 PBS (Vehicle Control) 73.4 ± 13.5mAb17664D (CD3 Binding Control-G) - 4 mg/kg 50.0 ± 6.6  REGN4460 (CD3Binding Control-G20) - 4 mg/kg 45.7 ± 6.1  REGN5458 (BCMA × CD3-G) - 4mg/kg 0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 1.0 1.0 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg 18.3 ± 5.0  REGN5459 (BCMA × CD3-G20) - 4 mg/kg 0.6± 0.6 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg 37.0 ± 5.7  Average Tumor Size (mm3) ± SEM on Day10 PBS (Vehicle Control) 249.9 ± 47.6  mAb17664D (CD3 BindingControl-G) - 4 mg/kg 125.0 ± 6.8  REGN4460 (CD3 Binding Control-G20) - 4mg/kg 173.9 ± 99   REGN5458 (BCMA × CD3-G) - 4 mg/kg 0 ± 0 REGN5458(BCMA × CD3-G) - 0.4 mg/kg 0 ± 0 REGN5458 (BCMA × CD3-G) - 0.04 mg/kg73.9 ± 25.7 REGN5459 (BCMA × CD3-G20) - 4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg  104 ±23.0 Average Tumor Size (mm3) ± SEM on Day 14 PBS (Vehicle Control)677.0 ± 62.7  mAb17664D (CD3 Binding Control-G) - 4 mg/kg 530.0 ± 44.6 REGN4460 (CD3 Binding Control-G20) - 4 mg/kg 549.1 ± 59.2  REGN5458(BCMA × CD3-G) - 4 mg/kg 0 ± 0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 0 ± 0REGN5458 (BCMA × CD3-G) - 0.04 mg/kg 255.4 ± 79.7  REGN5459 (BCMA ×CD3-G20) - 4 mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg 356.7 ± 84.6  Average Tumor Size(mm3) ± SEM on Day 17 PBS (Vehicle Control) 1349.5 ± 149.7  mAb17664D(CD3 Binding Control-G) - 4 mg/kg 935.3 ± 71.3  REGN4460 (CD3 BindingControl-G20) - 4 mg/kg 1027.1 ± 86.6  REGN5458 (BCMA × CD3-G) - 4 mg/kg14.5 ± 7.3  REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 1.7 ± 1.7 REGN5458 (BCMA× CD3-G) - 0.04 mg/kg 494.3 ± 144.3 REGN5459 (BCMA × CD3-G20) - 4 mg/kg0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg 645.6 ± 140.9 Average Tumor Size (mm3) ± SEM onDay 21 PBS (Vehicle Control) 2990.9 ± 291.7  mAb17664D (CD3 BindingControl-G) - 4 mg/kg 2249.6 ± 113.5  REGN4460 (CD3 BindingControl-G20) - 4 mg/kg 2473.4 ± 170.3  REGN5458 (BCMA × CD3-G) - 4 mg/kg102.7 ± 66.2  REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 5.3 ± 5.3 REGN5458(BCMA × CD3-G) - 0.04 mg/kg 1373.0 ± 366.6  REGN5459 (BCMA × CD3-G20) -4 mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA× CD3-G20) - 0.04 mg/kg 1442.4 ± 310.7  Average Tumor Size (mm3) ± SEMon Day 23 PBS (Vehicle Control) 4155.1 ± 401.8  mAb17664D (CD3 BindingControl-G) - 4 mg/kg 3288.4 ± 204.6  REGN4460 (CD3 BindingControl-G20) - 4 mg/kg 3592.7 ± 224.2  REGN5458 (BCMA × CD3-G) - 4 mg/kg193.3 ± 117.7 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 9.7 ± 9.7 REGN5458(BCMA × CD3-G) - 0.04 mg/kg 1882.3 ± 551.5  REGN5459 (BCMA × CD3-G20) -4 mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA× CD3-G20) - 0.04 mg/kg 2124.4 ± 444.1  Average Tumor Size (mm3) ± SEMon Day 28 PBS (Vehicle Control) Animals Euthanized mAb17664D (CD3Binding Control-G) - 4 mg/kg Animals Euthanized REGN4460 (CD3 BindingControl-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA × CD3-G) - 4mg/kg 627.4 ± 318.1 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 47.4 ± 47.4REGN5458 (BCMA × CD3-G) - 0.04 mg/kg 2542.5 ± 613.3  REGN5459 (BCMA ×CD3-G20) - 4 mg/kg 1.9 ± 1.9 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg 1939.3 ± 840.6  Average TumorSize (mm3) ± SEM on Day 31 PBS (Vehicle Control) Animals EuthanizedmAb17664D (CD3 Binding Control-G) - 4 mg/kg Animals Euthanized REGN4460(CD3 Binding Control-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA ×CD3-G) - 4 mg/kg 1018.5 ± 498.3  REGN5458 (BCMA × CD3-G) - 0.4 mg/kg104.7 ± 92.6  REGN5458 (BCMA × CD3-G) - 0.04 mg/kg 2906.1 ± 532.6 REGN5459 (BCMA × CD3-G20) - 4 mg/kg 3.8 ± 3.0 REGN5459 (BCMA ×CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) - 0.04 mg/kg 2688.7± 1176.6 Average Tumor Size (mm3) ± SEM on Day 35 PBS (Vehicle Control)Animals Euthanized mAb17664D (CD3 Binding Control-G) - 4 mg/kg AnimalsEuthanized REGN4460 (CD3 Binding Control-G20) - 4 mg/kg AnimalsEuthanized REGN5458 (BCMA × CD3-G) - 4 mg/kg 1342.9 ± 629.6  REGN5458(BCMA × CD3-G) - 0.4 mg/kg 375.1 ± 307.5 REGN5458 (BCMA × CD3-G) - 0.04mg/kg 3538.0 ± 0.0   REGN5459 (BCMA × CD3-G20) - 4 mg/kg 9.3 ± 7.5REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA × CD3-G20) -0.04 mg/kg 612.1 ± 0    Average Tumor Size (mm3) ± SEM on Day 42 PBS(Vehicle Control) Animals Euthanized mAb17664D (CD3 Binding Control-G) -4 mg/kg Animals Euthanized REGN4460 (CD3 Binding Control-G20) - 4 mg/kgAnimals Euthanized REGN5458 (BCMA × CD3-G) - 4 mg/kg 2363.0 ± 890.2 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg 968.8 ± 689.2 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg Animals Euthanized REGN5459 (BCMA × CD3-G20) - 4mg/kg 12.8 ± 12.8 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459(BCMA × CD3-G20) - 0.04 mg/kg Animals Euthanized Average Tumor Size(mm3) ± SEM on Day 49 PBS (Vehicle Control) Animals Euthanized mAb17664D(CD3 Binding Control-G) - 4 mg/kg Animals Euthanized REGN4460 (CD3Binding Control-G20) - 4 mg/kg Animals Euthanized REGN5458 (BCMA ×CD3-G) - 4 mg/kg 1683.5 ± 1683.5 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg NoRecording REGN5458 (BCMA × CD3-G) - 0.04 mg/kg Animals EuthanizedREGN5459 (BCMA × CD3-G20) - 4 mg/kg No Recording REGN5459 (BCMA ×CD3-G20) - 0.4 mg/kg No Recording REGN5459 (BCMA × CD3-G20) - 0.04 mg/kgAnimals Euthanized Average Tumor Size (mm3) ± SEM on Day 56 PBS (VehicleControl) Animals Euthanized mAb17664D (CD3 Binding Control-G) - 4 mg/kgAnimals Euthanized REGN4460 (CD3 Binding Control-G20) - 4 mg/kg AnimalsEuthanized REGN5458 (BCMA × CD3-G) - 4 mg/kg 3108.1 ± 3108.1 REGN5458(BCMA × CD3-G) - 0.4 mg/kg 1742.4 ± 635.2  REGN5458 (BCMA × CD3-G) -0.04 mg/kg Animals Euthanized REGN5459 (BCMA × CD3-G20) - 4 mg/kg 17.2 ±17.2 REGN5459 (BCMA × CD3-G20) - 0.4 mg/kg 0 ± 0 REGN5459 (BCMA ×CD3-G20) - 0.04 mg/kg Animals Euthanized

Example 14: Anti-BCMA x Anti-CD3 Bispecific Antibodies Delay Growth ofBCMA-Expressing Tumors (MOLP-8) in a Xenographic In Vivo Tumor Model

To determine the in vivo efficacy of anti-BCMA x anti-CD3 bispecificantibodies (Abs), a xenogenic tumor study was performed. On day −11,immunodeficient NOD.Cg-Prkdc^(scid)II2rg^(tm1Wji)/SzJ (NSG) mice wereintraperitoneally injected with 4×10⁶ human peripheral blood mononuclearcells (PBMC) from a normal, healthy donor. On day 0, the mice wereintravenously administered 2×10⁶ BCMA⁺ MOLP-8 human multiple myelomatumor cells that were engineered to also express firefly luciferase(MOLP-8-luciferase cells). The mice (n=5 per group) were thenimmediately administered a CD3-binding control bispecific Ab (G;mAb17664D) at a dose of 4 mg/kg or a BCMAxCD3 (G; REGN5458) bispecificAb at a dose of 4 mg/kg. The mice were administered these Abs twice moreon days 3 and 7, for a total of three doses. Tumor growth was assessedover 48 days by measuring tumor bioluminescence (BLI) in anesthetizedanimals. As a positive control, a group of mice (n=5) was given onlyMOLP-8-luciferase cells, but not PBMC or antibody. In order to measurebackground BLI levels, a group of mice (n=5) were untreated and did notreceive tumors, PBMC, or antibody. While the BCMA⁺ MOLP-8-luciferasetumors grew progressively in the CD3-binding control-treated mice,BCMAxCD3 Ab treatment with REGN5458 delayed the growth of tumors invivo.

Implantation and Measurement of Xenogenic Tumors:

On day −11, immunodeficient NOD.Cg-Prkdc^(scid)II2rg^(tm1Wji)/SzJ (NSG)mice were intraperitoneally injected with 5×10⁶ human PBMC from anormal, healthy donor. On day 0, the mice were intravenouslyadministered 2×10⁶ BCMA⁺ MOLP-8-luciferase cells. The mice (n=5 pergroup) were then immediately administered a CD3-binding controlbispecific Ab (G; mAb17664D) at a dose of 4 mg/kg or a BCMAxCD3 (G;REGN5458) bispecific Ab at a dose of 4 mg/kg. The mice were administeredthese Abs twice more on days 3 and 7, for a total of three doses. Tumorgrowth was assessed over 48 days by measuring tumor BLI in anesthetizedanimals. As a positive control, a group of mice (n=5) was given onlyMOLP-8-luciferase cells, but not PBMC or antibody. In order to measurebackground BLI levels, a group of mice (n=5) were untreated and did notreceive tumors, PBMC, or antibody.

Measurement of Xenogenic Tumor Growth:

BLI imaging was used to measure tumor burden. Mice were injected IP with150 mg/kg of the luciferase substrate D-luciferin suspended in PBS. Fiveminutes after this injection, BLI imaging of the mice was performedunder isoflurane anesthesia using the Xenogen IVIS system. Imageacquisition was carried out with the field of view at D, subject heightof 1.5 cm, and medium binning level with automatic exposure timedetermined by the Living Image Software. BLI signals were extractedusing Living Image software: regions of interest were drawn around eachtumor mass and photon intensities were recorded as p/s/cm2/sr.

Anti-BCMA x anti-CD3 bispecific antibody REGN5458 delayed the growth ofBCMA⁺ MOLP-8-luciferase tumors in this xenogenic in vivo tumor model.Results are shown in Table 22, below.

TABLE 22 Average Tumor Size (by radiance) at Various Time PointsAntibody Treatment Radiance [p/s/cm2²/sr] 8 days post- implantation(mean ± SEM) No tumor (background BLI) 4.93E+05 ± 1.66E+04 NoPBMC/Antibody (positive control) 5.73E+05 ± 5.27E+04 mAb17664D (CD3Binding Control-G) - 4 mg/kg 6.08E+05 ± 5.16E+04 REGN5458 (BCMA ×CD3-G) - 4 mg/kg 5.66E+05 ± 1.97E+04 Radiance [p/s/cm2²/sr] 15 dayspost- implantation (mean ± SEM) No tumor (background BLI) 5.37E+05 ±1.46E+04 No PBMC/Antibody (positive control) 1.24E+06 ± 9.67E+04mAb17664D (CD3 Binding Control-G) - 4 mg/kg 1.61E+06 ± 9.64E+04 REGN5458(BCMA × CD3-G) - 4 mg/kg 5.28E+05 ± 4.13E+04 Radiance [p/s/cm2²/sr] 22days post- implantation (mean ± SEM) No tumor (background BLI) 7.00E+05± 1.03E+04 No PBMC/Antibody (positive control) 1.23E+07 ± 1.02E+06mAb17664D (CD3 Binding Control-G) - 4 mg/kg 1.98E+07 ± 8.86E+06 REGN5458(BCMA × CD3-G) - 4 mg/kg 1.08E+06 ± 1.71E+05 Radiance [p/s/cm2²/sr] 24days post- implantation (mean ± SEM) No tumor (background BLI) 5.24E+05± 1.86E+04 No PBMC/Antibody (positive control) 1.56E+07 ± 1.29E+06mAb17664D (CD3 Binding Control-G) - 4 mg/kg 5.26E+07 ± 1.91E+07 REGN5458(BCMA × CD3-G) - 4 mg/kg 1.02E+06 ± 1.99E+05 Radiance [p/s/cm2²/sr] 28days post- implantation (mean ± SEM) No tumor (background BLI) 7.09E+05± 2.28E+04 No PBMC/Antibody (positive control) 3.01E+07 ± 4.78E+06mAb17664D (CD3 Binding Control-G) - 4 mg/kg 5.69E+07 ± 2.77E+07 REGN5458(BCMA × CD3-G) - 4 mg/kg 3.56E+06 ± 6.34E+05 Radiance [p/s/cm2²/sr] 30days post- implantation (mean ± SEM) No tumor (background BLI) 6.44E+05± 4.56E+04 No PBMC/Antibody (positive control) Animals EuthanizedmAb17664D (CD3 Binding Control-G) - 4 mg/kg Animals Euthanized REGN5458(BCMA × CD3-G) - 4 mg/kg 6.92E+06 ± 1.40E+06 Radiance [p/s/cm2²/sr] 34days post- implantation (mean ± SEM) No tumor (background BLI) 7.78E+05± 3.02E+04 No PBMC/Antibody (positive control) Animals EuthanizedmAb17664D (CD3 Binding Control-G) - 4 mg/kg Animals Euthanized REGN5458(BCMA × CD3-G) - 4 mg/kg 2.65E+07 ± 1.36E+07 Radiance [p/s/cm2²/sr] 37days post- implantation (mean ± SEM) No tumor (background BLI) 7.59E+05± 2.96E+04 No PBMC/Antibody (positive control) Animals EuthanizedmAb17664D (CD3 Binding Control-G) - 4 mg/kg Animals Euthanized REGN5458(BCMA × CD3-G) - 4 mg/kg 4.52E+07 ± 1.40E+07 Radiance [p/s/cm2²/sr] 43days post- implantation (mean ± SEM) No tumor (background BLI) 6.26E+05± 4.18E+04 No PBMC/Antibody (positive control) Animals EuthanizedmAb17664D (CD3 Binding Control-G) - 4 mg/kg Animals Euthanized REGN5458(BCMA × CD3-G) - 4 mg/kg 1.06E+08 ± 3.43E+07 Radiance [p/s/cm2²/sr] 48days post- implantation (mean ± SEM) No tumor (background BLI) 8.24E+05± 1.73E+04 No PBMC/Antibody (positive control) Animals EuthanizedmAb17664D (CD3 Binding Control-G) - 4 mg/kg Animals Euthanized REGN5458(BCMA × CD3-G) - 4 mg/kg 3.22E+08 ± 1.27E+08

Example 15: Anti-BCMA x Anti-CD3 Bispecific Antibodies Reduce Tumor(OPM-2) Burdens to Background Levels In Vivo

To determine the in vivo efficacy of anti-BCMA x anti-CD3 bispecificantibodies (Abs), a xenogenic tumor study was performed. On day 0,immunodeficient NOD.Cg-Prkdc^(scid)II2rg^(tm1Wji)/SzJ (NSG) mice wereintravenously administered 2×10⁶ BCMA⁺ OPM-2 human multiple myelomatumor cells that were engineered to also express firefly luciferase(OPM-2-luciferase cells). On day 10, the mice were intraperitoneallyinjected with 4×10⁶ human peripheral blood mononuclear cells (PBMC) froma normal, healthy donor. On day 21, the mice (n=5 per group) wereadministered a CD3-binding control bispecific Ab (G; mAb17664D) at adose of 0.4 mg/kg, a BCMAxCD3 (G; REGN5458) bispecific Ab at 0.4 mg/kg,or a BCMAxCD3 (G20; REGN5459) bispecific Ab at 0.4 mg/kg. The mice wereadministered these Abs twice more on days 25 and 28, for a total ofthree doses. Tumor growth was assessed through day 61 by measuring tumorbioluminescence (BLI) in anesthetized animals. As a positive control, agroup of mice (n=5) was given only OPM-2-luciferase cells, but not PBMCor antibody. In order to measure background BLI levels, a group of mice(n=5) were untreated and did not receive tumors, PBMC, or antibody.While the BCMA⁺ OPM-2-luciferase tumors grew progressively in theCD3-binding control-treated mice, BCMAxCD3 Ab treatment with REGN5458and REGN5459 reduced tumor burdens to background levels in the majorityof animals.

Implantation and Measurement of Xenogenic Tumors:

On day 0, immunodeficient NOD.Cg-Prkdc^(scid)II2rg^(tm1Wji)/SzJ (NSG)mice were intravenously administered 2×10⁶ BCMA⁺ OPM-2 human multiplemyeloma tumor cells that were engineered to also express fireflyluciferase (OPM-2-luciferase cells). On day 10, the mice wereintraperitoneally injected with 4×10⁶ human peripheral blood mononuclearcells (PBMC) from a normal, healthy donor. On day 21, the mice (n=5 pergroup) were administered a CD3-binding control bispecific Ab (G;mAb17664D) at a dose of 0.4 mg/kg, a BCMAxCD3 (G; REGN5458) bispecificAb at 0.4 mg/kg, or a BCMAxCD3 (G20; REGN5459) bispecific Ab at 0.4mg/kg. The mice were administered these Abs twice more on days 25 and28, for a total of three doses. Tumor growth was assessed through day 61by measuring tumor bioluminescence (BLI) in anesthetized animals. As apositive control, a group of mice (n=5) was given only OPM-2-luciferasecells, but not PBMC or antibody. In order to measure background BLIlevels, a group of mice (n=5) were untreated and did not receive tumors,PBMC, or antibody.

Measurement of Xenogenic Tumor Growth:

BLI imaging was used to measure tumor burden. Mice were injected IP with150 mg/kg of the luciferase substrate D-luciferin suspended in PBS. Fiveminutes after this injection, BLI imaging of the mice was performedunder isoflurane anesthesia using the Xenogen IVIS system. Imageacquisition was carried out with the field of view at D, subject heightof 1.5 cm, and medium binning level with automatic exposure timedetermined by the Living Image Software. BLI signals were extractedusing Living Image software: regions of interest were drawn around eachtumor mass and photon intensities were recorded as p/s/cm2/sr.

While the BCMA⁺ OPM-2-luciferase tumors grew progressively in theCD3-binding control-treated mice, BCMAxCD3 Ab treatment with REGN5458and REGN5459 reduced tumor burdens to background levels in the majorityof animals. Results are shown in Table 23, below, and illustrated inFIG. 7.

TABLE 23 Average Tumor Size (by radiance) at Various Time PointsAntibody Treatment Radiance [p/s/cm2²/sr] 5 days post- implantation(mean ± SEM) No tumor (background BLI) 6.22E+05 ± 2.77E+04 NoPBMC/Antibody (positive control) 5.62E+05 ± 2.75E+04 mAb17664D (CD3Binding Control-G) - 0.4 5.73E+05 ± 3.02E+04 mg/kg REGN5458 (BCMA ×CD3-G) - 0.4 mg/kg 5.87E+05 ± 2.40E+04 REGN5459 (BCMA × CD3-G20) - 0.4mg/kg 5.09E+05 ± 3.56E+04 Radiance [p/s/cm2²/sr] 11 days post-implantation (mean ± SEM) No tumor (background BLI) 6.90E+05 ± 3.64E+04No PBMC/Antibody (positive control) 6.22E+05 ± 3.34E+04 mAb17664D (CD3Binding Control-G) - 0.4 6.25E+05 ± 3.80E+04 mg/kg REGN5458 (BCMA ×CD3-G) - 0.4 mg/kg 6.19E+05 ± 4.39E+04 REGN5459 (BCMA × CD3-G20) - 0.4mg/kg 6.45E+05 ± 2.39E+04 Radiance [p/s/cm2²/sr] 20 days post-implantation (mean ± SEM) No tumor (background BLI) 7.59E+05 ± 5.82E+04No PBMC/Antibody (positive control) 2.32E+06 ± 2.94E+05 mAb17664D (CD3Binding Control-G) - 0.4 2.36E+06 ± 5.46E+05 mg/kg REGN5458 (BCMA ×CD3-G) - 0.4 mg/kg 1.81E+06 ± 2.37E+05 REGN5459 (BCMA × CD3-G20) - 0.4mg/kg 2.13E+06 ± 1.69E+05 Radiance [p/s/cm2²/sr] 26 days post-implantation (mean ± SEM) No tumor (background BLI) 5.51E+05 ± 2.51E+04No PBMC/Antibody (positive control) 5.96E+06 ± 8.74E+05 mAb17664D (CD3Binding Control-G) - 0.4 6.05E+06 ± 1.32E+06 mg/kg REGN5458 (BCMA ×CD3-G) - 0.4 mg/kg 1.73E+06 ± 8.69E+05 REGN5459 (BCMA × CD3-G20) - 0.4mg/kg 1.28E+06 ± 7.36E+05 Radiance [p/s/cm2²/sr] 31 days post-implantation (mean ± SEM) No tumor (background BLI) 6.62E+05 ± 3.35E+04No PBMC/Antibody (positive control) 1.58E+07 ± 4.84E+06 mAb17664D (CD3Binding Control-G) - 0.4 1.35E+07 ± 2.35E+06 mg/kg REGN5458 (BCMA ×CD3-G) - 0.4 mg/kg 3.50E+06 ± 2.42E+06 REGN5459 (BCMA × CD3-G20) - 0.4mg/kg 1.98E+06 ± 1.36E+06 Radiance [p/s/cm2²/sr] 34 days post-implantation (mean ± SEM) No tumor (background BLI) 4.57E+05 ± 1.04E+04No PBMC/Antibody (positive control) 3.36E+07 ± 1.27E+07 mAb17664D (CD3Binding Control-G) - 0.4 2.35E+07 ± 5.72E+06 mg/kg REGN5458 (BCMA ×CD3-G) - 0.4 mg/kg 4.85E+06 ± 3.24E+06 REGN5459 (BCMA × CD3-G20) - 0.4mg/kg 4.24E+06 ± 3.69E+06 Radiance [p/s/cm2²/sr] 38 days post-implantation (mean ± SEM) No tumor (background BLI) 6.60E+05 ± 3.13E+04No PBMC/Antibody (positive control) 3.91E+07 ± 6.87E+06 mAb17664D (CD3Binding Control-G) - 0.4 4.84E+07 ± 1.65E+07 mg/kg REGN5458 (BCMA ×CD3-G) - 0.4 mg/kg 5.30E+06 ± 3.44E+06 REGN5459 (BCMA × CD3-G20) - 0.4mg/kg 3.21E+06 ± 2.52E+06 Radiance [p/s/cm2²/sr] 40 days post-implantation (mean ± SEM) No tumor (background BLI) 5.39E+05 ± 9.67E+03No PBMC/Antibody (positive control) Animals euthanized mAb17664D (CD3Binding Control-G) - 0.4 Animals euthanized mg/kg REGN5458 (BCMA ×CD3-G) - 0.4 mg/kg 5.06E+06 ± 3.36E+06 REGN5459 (BCMA × CD3-G20) - 0.4mg/kg 3.84E+06 ± 3.34E+06 Radiance [p/s/cm2²/sr] 47 days post-implantation (mean ± SEM) No tumor (background BLI) 7.73E+05 ± 1.91E+04No PBMC/Antibody (positive control) Animals euthanized mAb17664D (CD3Binding Control-G) - 0.4 Animals euthanized mg/kg REGN5458 (BCMA ×CD3-G) - 0.4 mg/kg 7.76E+05 ± 7.85E+04 REGN5459 (BCMA × CD3-G20) - 0.4mg/kg 7.34E+05 ± 2.62E+04 Radiance [p/s/cm2²/sr] 54 days post-implantation (mean ± SEM) No tumor (background BLI) 7.49E+05 ± 1.95E+04No PBMC/Antibody (positive control) Animals euthanized mAb17664D (CD3Binding Control-G) - 0.4 Animals euthanized mg/kg REGN5458 (BCMA ×CD3-G) - 0.4 mg/kg 5.78E+05 ± 1.15E+05 REGN5459 (BCMA × CD3-G20) - 0.4mg/kg 6.41E+05 ± 5.96E+04 Radiance [p/s/cm2²/sr] 61 days post-implantation (mean ± SEM) No tumor (background BLI) 6.18E+05 ± 2.77E+04No PBMC/Antibody (positive control) Animals euthanized mAb17664D (CD3Binding Control-G) - 0.4 Animals euthanized mg/kg REGN5458 (BCMA ×CD3-G) - 0.4 mg/kg 5.23E+05 ± 4.10E+04 REGN5459 (BCMA × CD3-G20) - 0.4mg/kg 6.03E+05 ± 5.29E+04

Example 16: BCMAxCD3 Bispecific Antibodies Suppress Growth of SyngenicTumors In Vivo in a Dose-Dependent Manner

To determine the in vivo efficacy of anti-BCMA x anti-CD3 bispecificantibodies (Abs), a syngenic tumor study was performed in miceexpressing human CD3. C57BL/6 mice that express human CD3deg in place ofmurine CD3deg (CD3-humanized mice) were subcutaneously implanted witheither 0.5×10⁶ B16 melanoma cells that have been engineered to expressfull-length human BCMA (B16/BCMA cells) or 1×10⁶ MC38 colon carcinomacells that have been engineered to express full-length human BCMA(MC38/BCMA). The mice (n=7 per group) were then immediately administereda CD3-binding control bispecific Ab (G; mAb17664D) at a dose of 0.4mg/kg or a BCMAxCD3 (G; REGN5458) bispecific Ab at doses of either 0.4mg/kg or 0.04 mg/kg. The mice were administered these Abs twice more ondays 4 and 7 for a total of three doses, and tumor growth was assessedthroughout the experiment. While the B16/BCMA tumors and the MC38/BCMAtumors grew in the CD3-binding control-treated mice, BCMAxCD3 REGN5458was able to suppress the growth of both tumor lines in a dose-dependentmanner in vivo.

Implantation and Measurement of Syngenic Tumors:

C57BL/6 mice that express human CD3deg in place of murine CD3deg(CD3-humanized mice) were subcutaneously implanted with either 0.5×10⁶B16F10 melanoma cells that have been engineered to express full-lengthhuman BCMA (B16/BCMA cells) or 1×10⁶ MC38 colon carcinoma cells thathave been engineered to express full-length human BCMA (MC38/BCMA). Themice (n=7 per group) were then immediately administered a CD3-bindingcontrol bispecific Ab (G; mAb17664D) at a dose of 0.4 mg/kg or aBCMAxCD3 (G; REGN5458) bispecific Ab at doses of either 0.4 mg/kg or0.04 mg/kg. The mice were administered these Abs twice more on days 4and 7 for a total of three doses, and tumor growth was assessedthroughout the experiment.

Calculation of Syngenic Tumor Growth and Inhibition:

In order to determine tumor volume by external caliper, the greatestlongitudinal diameter (length in mm) and the greatest transversediameter (width in mm) were determined. Tumor volumes based on calipermeasurements were calculated by the formula: Volume(mm³)=(length×width^(z))/2.

While the B16/BCMA tumors and the MC38/BCMA tumors grew in theCD3-binding control-treated mice, BCMAxCD3 REGN5458 was able to suppressthe growth of both tumor lines in a dose-dependent manner in vivo.Results are shown in Table 24, below.

TABLE 24 Average Tumor Size at Various Time Points Antibody TreatmentAverage Tumor Size (mm3) ± SEM on Day 5 B16/BCMA Tumor 25.6 ± 2.7 mAb17664D (CD3 Binding Control-G) - 0.4 mg/kg B16/BCMA Tumor 0.0 ± 0.0REGN5458 (BCMA × CD3-G) - 0.4 mg/kg B16/BCMA Tumor 3.3 ± 2.2 REGN5458(BCMA × CD3-G) - 0.04 mg/kg MC38/BCMA Tumor 29.3 ± 4.4  mAb17664D (CD3Binding Control-G) - 0.4 mg/kg MC38/BCMA Tumor 1.4 ± 1.4 REGN5458 (BCMA× CD3-G) - 0.4 mg/kg MC38/BCMA Tumor 11.9 ± 2.9  REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg Average Tumor Size (mm3) ± SEM on Day 10 B16/BCMATumor 179.2 ± 30.6  mAb17664D (CD3 Binding Control-G) - 0.4 mg/kgB16/BCMA Tumor 0.0 ± 0.0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg B16/BCMATumor 15.4 ± 12.5 REGN5458 (BCMA × CD3-G) - 0.04 mg/kg MC38/BCMA Tumor123.1 ± 14.6  mAb17664D (CD3 Binding Control-G) - 0.4 mg/kg MC38/BCMATumor 0.0 ± 0.0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg MC38/BCMA Tumor 66.7± 22.5 REGN5458 (BCMA × CD3-G) - 0.04 mg/kg Average Tumor Size (mm3) ±SEM on Day 14 B16/BCMA Tumor 763.1 ± 156.2 mAb17664D (CD3 BindingControl-G) - 0.4 mg/kg B16/BCMA Tumor 8.1 ± 4.4 REGN5458 (BCMA ×CD3-G) - 0.4 mg/kg B16/BCMA Tumor 81.4 ± 49.2 REGN5458 (BCMA × CD3-G) -0.04 mg/kg MC38/BCMA Tumor 477.1 ± 77.1  mAb17664D (CD3 BindingControl-G) - 0.4 mg/kg MC38/BCMA Tumor 2.9 ± 2.9 REGN5458 (BCMA ×CD3-G) - 0.4 mg/kg MC38/BCMA Tumor 273.3 ± 115.3 REGN5458 (BCMA ×CD3-G) - 0.04 mg/kg Average Tumor Size (mm3) ± SEM on Day 18 B16/BCMATumor 2068.9 ± 357.7  mAb17664D (CD3 Binding Control-G) - 0.4 mg/kgB16/BCMA Tumor 47.1 ± 17.0 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg B16/BCMATumor 127.2 ± 63.9  REGN5458 (BCMA × CD3-G) - 0.04 mg/kg MC38/BCMA Tumor1432.5 ± 231.6  mAb17664D (CD3 Binding Control-G) - 0.4 mg/kg MC38/BCMATumor 7.5 ± 7.5 REGN5458 (BCMA × CD3-G) - 0.4 mg/kg MC38/BCMA Tumor641.5 ± 309.8 REGN5458 (BCMA × CD3-G) - 0.04 mg/kg

Example 17: Epitope Mapping of REGN5458 Binding to BCMA by HydrogenDeuterium Exchange

H/D exchange epitope mapping with mass spectrometry (HDX-MS) wasperformed to determine the amino acid residues of BCMA (recombinanthuman BCMA, amino acid sequence of SEQ ID NO: 115) interacting withREGN5458 (BCMA x CD3 bispecific antibody). A general description of theH/D exchange method is set forth in e.g., Ehring (1999) AnalyticalBiochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem.73:256A-265A.

The HDX-MS experiments were performed on an integrated HDX/MS platform,consisting of a Leaptec HDX PAL system for the deuterium labeling andquenching, a Waters Acquity M-Class (Auxiliary solvent manager) for thesample digestion and loading, a Waters Acquity M-Class (μBinary solventmanager) for the analytical gradient, and Thermo Q Exactive HF massspectrometer for peptide mass measurement.

The labeling solution was prepared as PBS buffer in D₂O at pD 7.0 (10 mMphosphate buffer, 140 mM NaCl, and 3 mM KCl, equivalent to pH 7.4 at 25°C.). For deuterium labeling, 10 μL of hBCMA.hFc (REGN2746, 54.5 μM; SEQID NO: 120 or hBCMA.hFc premixed with REGN5458 in 1:2 molar ratio (Ag-Abcomplex) was incubated at 20° C. with 90 μL D₂O labeling solution forvarious time-points in duplicates (e.g., Undeuterated control=0 second;deuterium-labeled for 5 minutes and 10 minutes). The deuterationreaction was quenched by adding 100 μL of pre-chilled quench buffer (0.5M TCEP-HCl, 8 M urea and 1% formic acid) to each sample for a 5-minuteincubation at 20° C. The quenched sample was then injected into a WatersHDX Manager for online pepsin/protease XIII digestion. The digestedpeptides were separated by a C8 column (1.0 mm×50 mm, NovaBioassays)with a 13-minute gradient from 10%-32% B (mobile phase A: 0.5% formicacid in water, mobile phase B: 0.1% formic acid in acetonitrile). Theeluted peptides were analyzed by Q Exactive HF mass spectrometry inLC-MS/MS or LC-MS mode.

The LC-MS/MS data of undeuterated BCMA sample were searched against adatabase including BCMA and its randomized sequence using Byonic searchengine (Protein Metrics). The search parameters (in ELN) were set asdefault using non-specific enzymatic digestion and human glycosylationas common variable modification. The list of identified peptides wasthen imported into the HDX Workbench software (version 3.3) to calculatethe deuterium uptake of each peptide detected by LC-MS from alldeuterated samples. For a given peptide, the centroid mass(intensity-weighted average mass) at each time point was used tocalculate the deuterium uptake (D) and percentage of deuterium uptake (%D):

 Deuterium  Uptake  (D-uptake) = Average  Mass(deuterated) − Average  Mass  (undeuterated)${{Percentage}\mspace{14mu} {of}\mspace{14mu} {deuterium}\mspace{14mu} {uptake}\mspace{14mu} \left( {\% \mspace{11mu} D} \right)} = \frac{D\text{-}{uptake}\mspace{14mu} {for}\mspace{14mu} {peptide}\mspace{14mu} {at}\mspace{14mu} {each}\mspace{14mu} {time}\mspace{14mu} {point}\mspace{14mu} \times 100\%}{{Maximum}\mspace{14mu} D\text{-}{uptake}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {{peptide}{\mspace{11mu} \;}\left( {{defined}\mspace{14mu} {in}\mspace{14mu} {ELN}} \right)}}$

A total of 8 peptides from hBCMA.hFc were identified from both hBCMA.hFcalone and hBCMA.hFc in complex with REGN5458 samples, representing 100%sequence coverage of hBCMA. The averaged standard deviation (SD) of allpeptides was evaluated to be 1.4% (detailed calculations were defined inELN and Pascal, B D et al (2012) Journal of the American Society forMass Spectrometry 23(9):1512-1521). Therefore, any peptide whichexhibited a differential percent D-uptake values above 4.2% (3-fold ofthe averaged SD) was defined as significantly protected. For hBCMA.hFc,peptides corresponding to amino acids 1-43 of SEQ ID NO: 106(MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNA; SEQ ID NO: 121) weresignificantly protected by REGN5458. Protection of these residues byREGN5458 was confirmed using hBCMA.mmH (REGN2744, amino acid sequence ofSEQ ID NO: 106).

TABLE 25 Selected BCMA.hFc peptides with significant protection uponbinding to REGN5458 5 min 10 min REGN2746 REGN2746 + + REGN2746 REGN5458REGN2746 REGN5458 BCMA Centroid Centroid Centroid Centroid -hFc ResiduesMH⁺ MH⁺ ΔD MH⁺ MH⁺ ΔD Δ % D 1-28 3217.16 3212.39 −4.77 3218.05 3212.62−5.43 −25.2 4-26 2582.03 2577.26 −4.77 2582.71 2577.45 −5.26 −31 27-43 1921.75 1920.69 −1.06 1922.1 1920.83 −1.27 −11.1

Example 18: FACS Binding Assay of BCMAxCD3 Bispecific Antibodies andAdditional BCMA Antibodies on Multiple Myeloma Cell Lines afterOvernight Incubation with Anti-BCMA Antibodies

Flow cytometric analysis was utilized to determine the impact ofovernight incubation of multiple myeloma cell lines with anti-BCMAantibodies on the level of surface BCMA. MM cell lines (H929, Molp8,U266 and MM1.S) were washed two times and cultured at 37° C. in R10media (RPMI+10% FBS+pen/strep/glut) containing 66.7 or 667 nM anti-BCMAantibodies, DAPT (a gamma-secretase inhibitor) or media only. After 18hours, wells were washed with cold FACS wash (PBS+1% filtered FBS) andresuspended in 667 nM of the same anti-BCMA antibody in cold stainbuffer (Miltenyi 130-091-221) for 30 minutes on ice. After incubation,the cells were washed twice with cold FACS wash (PBS+1% filtered FBS)and bound antibody was detected by incubating with the appropriateanti-human secondary antibody (anti-hIgG or anti-HIS) on ice for anadditional 30-45 minutes. After incubation, cells were washed,re-suspended in 200 μL cold PBS containing 1% filtered FBS and analyzedby flow cytometry on a BD FACS Canto II. Fold increase in staining wascalculated by dividing the MFI of stained cells previously incubatedovernight in BCMA abs or DAPT by the MFI of stained cells that wereincubated overnight in media only.

BCMA is rapidly cleaved from the surface of cells by the enzymegamma-secretase. Overnight incubation with the gamma-secretaseinhibitors, such as DAPT, prevents BCMA cleavage resulting in increasedlevels of BCMA on the cell surface. Tables 26-29 report the foldincrease in the median fluorescence intensity (MFI) of BCMA on cellsincubated overnight in anti-BCMA antibodies or DAPT compared to cellsincubated in media only. We observed that overnight incubation with DAPTincreased BCMA levels detected by anti-BCMA antibodies (BCMAxCD3bispecific R5458, the parental BCMA antibody mAb15281, and other inhouse BCMA antibodies) on H929, Molp8, U266 and MM.1S, 2.3-4 fold,2.4-8.6 fold, 5.3-9.0 fold, and 11.9 fold, respectively.

Of note, we also observed that overnight incubation of MM cell lineswith 66.7 or 667 nM REGN5458 or the parental bivalent anti-BCMA antibodymAb21581 similarly resulted in increased levels of surface BCMA detectedby FACS, suggesting that binding of anti-BCMA antibodies preventscleavage of BCMA by gamma-secretase. Antibody induced increases insurface BCMA differed by cell line, with greater fold increases on Molp8and MM1S cells compared to H929 or U266. The phenomenon was not limitedto REGN5458, as it was also observed with other in house BCMAantibodies.

TABLE 26 MFI fold change over cells incubated in media only (NCI-H929)67 nM 667 nM DAPT NCI-H929 Average n Average n Average n mAb21581 aBCMA(parent 1.2 5 1.4 3 3.5 6 to R5458) REGN5458 BCMA × CD3 2.0 3 3.0 1 4.03 mAb16749 aBCMA 1.0 2 0.8 1 2.3 3 mAb16711 aBCMA 2.8 2 2.1 1 3.8 3mAb16747 aBCMA 1.8 2 2.1 1 3.9 3 REGN960 scFv IsoC 1.0 2 1.1 1 1.1 3mAb11810 IgG1 IsoC 1.0 2 1.0 1 1.1 3 mAb11810 IgG4s IsoC 1.3 2 1.0 1 1.13

TABLE 27 MFI fold change over cells incubated in media only (Molp8) 67nM 667 nM DAPT Molp8 Average n Average n Average n mAb21581 aBCMA(parent 2.3 5 3.7 3 6.3 6 to R5458) REGN5458 BCMA × CD3 2.3 3 4.5 1 8.63 mAb16749 aBCMA 1.1 2 3.4 1 4.0 3 mAb16711 aBCMA 3.5 2 3.0 1 5.1 3mAb16747 aBCMA 2.2 2 0.6 1 6.2 3 REGN960 scFv IsoC 1.1 2 1.0 1 1.0 3mAb11810 IgG1 IsoC 1.0 2 1.3 1 1.1 3 mAb11810 IgG4s IsoC 0.9 2 1.2 1 1.03

TABLE 28 MFI fold change over cells incubated in media only (U266) 67 nM667 nM DAPT U266 Average n Average n Average n mAb21581 aBCMA (parent1.8 2 2.3 1 6.7 6 to R5458) REGN5458 BCMA × CD3 1.4 2 2.3 1 9.0 3mAb16749 aBCMA 1.3 2 1.2 1 5.3 3 mAb16711 aBCMA 2.2 2 2.2 1 7.2 3mAb16747 aBCMA 1.5 2 1.7 1 8.3 3 REGN960 scFv IsoC 1.0 2 1.0 1 1.0 3mAb11810 IgG1 IsoC 1.0 2 1.1 1 1.1 3 mAb11810 IgG4s IsoC 1.1 2 1.1 1 1.43

TABLE 29 MFI fold change over cells incubated in media only (MM1S) 67 nM667 nM DAPT MM1S Average n Average n Average n mAb21581 aBCMA (parent7.3 2 7.0 2 11.9 2 tomR5458)

Example 19: Autologous T Cell-Mediated Killing of Human and CynomolgusMonkey Plasma Cells in the Presence of BCMAxCD3 Bispecific Antibodies

The specific killing of enriched CD138⁺ human or cynomolgus monkeyplasma cells by unstimulated autologous T cells was assessed by flowcytometry. Human or cynomolgus bone marrow aspirates and blood wereprovided within 24 hours of harvest. CD13⁸ plasma cells were enrichedfrom bone marrow by positive selection using the EasySep Human CD13⁸Positive Selection kit according to the manufacturer's instructions.PBMC from whole blood were isolated by density separation. PBMC werelabeled with 1 μM of Vybrant CFDA-SE fluorescent tracking dye. Afterlabeling, 1×10⁴ enriched CD13⁸ plasma cells were plated in round-bottom96 well plates at an E:T ratio of 10:1 with Vybrant CFDA-SE labeled PBMCand serial dilutions of REGN5458, CD3-binding control bsAb, orBCMA-binding control mAb for 72 hours at 37° C. in complete media. Atthe end of the culture, surviving CD13⁸ plasma cells were analyzed byflow cytometry, utilizing fixable LIVE/DEAD dye and plasma cell specificcell surface markers. Percent viability was normalized to controlcondition (plasma cells in the presence of PBMC only). T cell activationwas assessed by flow cytometry. Activation is reported as the percentageof CD2⁺/CD4⁺ or CD2⁺/CD8⁺/CD16⁻ T cells expressing CD25. Percent T cellactivation was normalized to control condition (plasma cells in thepresence of PBMC only).

In vitro studies evaluated the effect of REGN5458 or negative controls(BCMA-binding control mAb or CD3-binding control bsAb) on primary humanand cynomolgus monkey T cell activation and cytotoxicity of autologousplasma cells. The EC₅₀ values for cytotoxicity and percent T cellactivation for each donor are summarized in Table 30.

REGN5458 mediated cytotoxicity of primary human plasma cells from donors1 and 2 in the presence autologous T cells in a concentration-dependentmanner with EC₅₀ values of 42.8 pM and 191 pM, respectively, andresulted in a maximum percent cytotoxicity of 91% and 89%, respectively.In parallel, REGN5458 mediated T cell activation in the presence ofhuman plasma cells from donors 1 and 2 in a concentration-dependentmanner with EC₅₀ values of 214 pM and 860 pM for CD8⁺ T cell activation,respectively, and maximum percent CD8⁺ T cell activation of 2% and 36%,respectively. Cytotoxicity of plasma cells in both donors and increasedCD8⁺ T cell activation in donor 2 only was observed at nanomolarconcentrations of CD3-binding control. No effect on cytotoxicity or Tcell activation was observed with BCMA-binding control at any of theconcentrations tested in either donor.

REGN5458 mediated cytotoxicity of primary cynomolgus plasma cells inboth donors in a concentration-dependent manner; an EC₅₀ of 1.31 nM wascalculated for donor 1, however an EC₅₀ could not be determined fordonor 2. In both donors, REGN5458 treatment resulted in increasedcytotoxicity of plasma cells (maximum percent cytotoxicity of 94% and91% for donors 1 and 2, respectively). In parallel, REGN5458 mediated Tcell activation in the presence of cynomolgus monkey plasma cells fromdonors 1 and 2 in a concentration-dependent manner with EC₅₀ values of28.1 nM and 18.1 nM for CD4⁺ T cell activation and 22.4 nM and 76.7 nMfor CD8⁺ T cell activation, respectively. The resulting maximum percentT cell activation was 9% and 16% for CD4⁺ T cells and 12% and 17% CD8⁺ Tcells for donors 1 and 2, respectively.

No target cell killing was observed with BCMA-binding control at anyconcentration tested in either of the cell lines evaluated. Some targetcell killing and T cell activation in the presence of plasma cells fromdonor 2 was observed with CD3-binding control at nanomolarconcentrations.

TABLE 30 EC₅₀ Values for Cytotoxicity and Percent T Cell Activation forEach Donor T Cell Activation (% CD25 Upregulation) Cell Lines CytotoxicKill CD4⁺ T cells CD8⁺ T cells Effector Max % % % Cells Target CellsEC₅₀ (M) Cytotoxicity EC₅₀ (M) Activation EC₅₀ (M) Activation PrimaryHuman Donor 1 4.28 × 10⁻¹¹ 91 NR NR 2.14 × 10⁻¹⁰ 2 Human T Plasma CellsCells^(a) Human Donor 2 1.91 × 10⁻¹⁰ 89 NR NR 8.60 × 10⁻¹⁰ 36 PlasmaCells Primary Cynomolgus Monkey 1.31 × 10⁻⁹  94   2.81 × 10⁻⁹ 9 2.24 ×10⁻⁹  12 Cynomolgus Donor 1 Plasma Cells Monkey T Cynomolgus Monkey ND91 ~1.81 × 10⁻⁸ 16 7.67 × 10⁻⁸  17 Cells^(a) Donor 2 Plasma Cells^(a)Autologous plasma cells were tested for each donor.

Example 20: Anti-BCMA x Anti-CD3 Bispecific Antibodies ActSynergistically with Anti-PD-1 Antibodies to Enhance Anti-Tumor EfficacyIn Vivo

To determine whether BCMAxCD3 bispecific antibodies (Abs) synergize withPD-1 blockade to provide superior anti-tumor efficacy in vivo, asyngenic tumor study was performed in mice expressing human CD3. Theresults demonstrate that combining REGN5458 plus PD-1 blockade providessuperior anti-tumor efficacy than either REGN5458 or PD-1 blockadealone.

Implantation and Measurement of Syngenic Tumors:

C57BL/6 mice that express human CD3deg in place of murine CD3deg(CD3-humanized mice) were subcutaneously implanted with 1×10⁶ MC38 coloncarcinoma cells that have been engineered to express full-length humanBCMA (MC38/BCMA). The tumors were allowed to establish for 3 days, atwhich time the mice (n=6 or 7 per group) were administered a CD3-bindingcontrol bispecific Ab (G; H4sH17664D) at a dose of 0.4 mg/kg or aBCMAxCD3 (G; REGN5458) bispecific Ab at doses of either 0.04 mg/kg or0.24 mg/kg, along with either a surrogate anti-mouse PD-1 antibody(Clone RPM1-14) at 4 mg/kg or an isotype control Ab (Clone 2A3) at 4mg/kg. The specific treatment groups are shown in Table 31, below.

TABLE 31 Treatment Groups Group Bispecific Treatment Antibody n 1H4SH17664D (0.24 mg/kg) Isotype (4 mg/kg) 7 2 H4SH17664D (0.24 mg/kg)RPM1-14 (4 mg/kg) 7 3 REGN5458 (0.04 mg/kg) Isotype (4 mg/kg) 7 4REGN5458 (0.04 mg/kg) RPM1-14 (4 mg/kg) 7 5 REGN5458 (0.24 mg/kg)Isotype (4 mg/kg) 6 6 REGN5458 (0.24 mg/kg) RPM1-14 (4 mg/kg) 6

The mice were administered these Abs twice more on days 7 and 11 for atotal of three doses, and tumor growth was assessed throughout theexperiment.

Calculation of Syngenic Tumor Growth and Inhibition:

In order to determine tumor volume by external caliper, the greatestlongitudinal diameter (length in mm) and the greatest transversediameter (width in mm) were determined. Tumor volumes based on calipermeasurements were calculated by the formula: Volume(mm³)=(length×width²)/2.

The results demonstrate that combining REGN5458 plus PD-1 blockadeprovides superior anti-tumor efficacy than either REGN5458 or PD-1blockade alone. In particular, the results demonstrate that at day 24(the last day for which data was collected for all treatment groups) thecombination of BCMAxCD3 bispecific antibody and anti-PD-1 antibodyproduced a statistically significant synergistic therapeutic effect inthe inhibition of tumor growth (Table 32, BCMAxCD3 at 0.04 mg/kg andanti-PD-1 at 4 mg/kg). Using a 2-way ANOVA test at day 24, p<0.0001between (i) REGN5458 (0.04 mg/kg)+Isotype and the combination ofREGN5458 (0.04 mg/kg)+anti-PD-1 antibody (Group 3 vs. Group 4), (ii)REGN5458 (0.24 mg/kg)+Isotype and the combination of REGN5458 (0.24mg/kg)+anti-PD-1 antibody (Group 5 vs. Group 6), (iii) anti-PD-1 and thecombination of REGN5458 (0.04 mg/kg)+anti-PD-1 antibody (Group 2 vs.Group 6). Using a 2-way ANOVA test at day 24, p=0.0005 between anti-PD-1and the combination of REGN5458 (0.04 mg/kg)+anti-PD-1 antibody (Group 2vs. Group 4). Increasing the dose of BCMAxCD3 bispecific antibody (0.24mg/kg) in combination with PD-1 blockade resulted in tumor inhibitioncomparable to the lower bispecific antibody dose plus PD-1 blockade inthis experiment. The demonstrated synergy with the lower dose bispecificantibody is advantageous because the use of a lower dose reduces therisk of any adverse side effects. Similarly, the combination of BCMAxCD3bispecific antibody and anti-PD-1 antibody showed a synergistictherapeutic effect at both doses of bispecific antibody (0.04 mg/kg and0.24 mg/kg) in the number of tumor-free mice at the end of theexperiment (day 28), as shown in Table 33.

TABLE 32 Average Tumor Size at Various Time Points Antibody TreatmentAverage Tumor Size (mm3) ± SEM  on Day 3 CD3-binding control H4SH17664D(0.24 16.30 ± 1.50 mg/kg) + Isotype (4 mg/kg) n = 7 CD3-binding controlH4SH17664D (0.24 14.34 ± 1.17 mg/kg) + PD-1-blocking RPM1-14 (4 mg/kg) n= 7 BCMA × CD3 REGN5458 (0.04 mg/kg) + 15.62 ± 1.61 Isotype (4 mg/kg) n= 7 BCMA × CD3 REGN5458 (0.04 mg/kg) + 19.20 ± 2.94 PD-1-blockingRPM1-14 (4 mg/kg) n = 7 BCMA × CD3 REGN5458 (0.24 mg/kg) + 13.13 ± 3.12Isotype (4 mg/kg) n = 6 BCMA × CD3 REGN5458 (0.24 mg/kg) + 20.41 ± 3.15PD-1-blocking RPM1-14 (4 mg/kg) n = 6 Average Tumor Size (mm3) ± SEM  onDay 7 CD3-binding control H4SH17664D (0.24 55.78 ± 6.61 mg/kg) + Isotype(4 mg/kg) n = 7 CD3-binding control H4SH17664D (0.24 43.59 ± 8.32mg/kg) + PD-1-blocking RPM1-14 (4 mg/kg) n = 7 BCMA × CD3 REGN5458 (0.04mg/kg) + 37.98 ± 3.93 Isotype (4 mg/kg) n = 7 BCMA × CD3 REGN5458 (0.04mg/kg) + 30.30 ± 6.47 PD-1-blocking RPM1-14 (4 mg/kg) n = 7 BCMA × CD3REGN5458 (0.24 mg/kg) + 29.27 ± 5.00 Isotype (4 mg/kg) n = 6 BCMA × CD3REGN5458 (0.24 mg/kg) + 29.18 ± 3.65 PD-1-blocking RPM1-14 (4 mg/kg) n =6 Average Tumor Size (mm3) ± SEM  on Day 11 CD3-binding controlH4SH17664D (0.24 145.74 ± 21.37 mg/kg) + Isotype (4 mg/kg) n = 7CD3-binding control H4SH17664D (0.24  45.33 ± 11.46 mg/kg) +PD-1-blocking RPM1-14 (4 mg/kg) n = 7 BCMA × CD3 REGN5458 (0.04 mg/kg) +112.53 ± 17.39 Isotype (4 mg/kg) n = 7 BCMA × CD3 REGN5458 (0.04mg/kg) +  8.81 ± 0.88 PD-1-blocking RPM1-14 (4 mg/kg) n = 7 BCMA × CD3REGN5458 (0.24 mg/kg) +  36.63 ± 14.89 Isotype (4 mg/kg) n = 6 BCMA ×CD3 REGN5458 (0.24 mg/kg) + 12.99 ± 4.35 PD-1-blocking RPM1-14 (4 mg/kg)n = 6 Average Tumor Size (mm3) ± SEM  on Day 14 CD3-binding controlH4SH17664D (0.24 414.28 ± 46.72 mg/kg) + Isotype (4 mg/kg) n = 7CD3-binding control H4SH17664D (0.24  49.50 ± 17.02 mg/kg) +PD-1-blocking RPM1-14 (4 mg/kg) n = 7 BCMA × CD3 REGN5458 (0.04 mg/kg) +438.16 ± 59.56 Isotype (4 mg/kg) n = 7 BCMA × CD3 REGN5458 (0.04mg/kg) +  6.86 ± 3.90 PD-1-blocking RPM1-14 (4 mg/kg) n = 7 BCMA × CD3REGN5458 (0.24 mg/kg) + 224.33 ± 47.04 Isotype (4 mg/kg) n = 6 BCMA ×CD3 REGN5458 (0.24 mg/kg) +  22.75 ± 17.62 PD-1-blocking RPM1-14 (4mg/kg) n = 6 Average Tumor Size (mm3) ± SEM  on Day 18 CD3-bindingcontrol H4SH17664D (0.24 1035.43 ± 123.41 mg/kg) + Isotype (4 mg/kg) n =6 CD3-binding control H4SH17664D (0.24 100.83 ± 41.62 mg/kg) +PD-1-blocking RPM1-14 (4 mg/kg) n = 7 BCMA × CD3 REGN5458 (0.04 mg/kg) +1040.12 ± 61.95  Isotype (4 mg/kg) n = 7 BCMA × CD3 REGN5458 (0.04mg/kg) +  7.81 ± 7.81 PD-1-blocking RPM1-14 (4 mg/kg) n = 7 BCMA × CD3REGN5458 (0.24 mg/kg) +  515.15 ± 115.38 Isotype (4 mg/kg) n = 6 BCMA ×CD3 REGN5458 (0.24 mg/kg) +  57.79 ± 43.62 PD-1-blocking RPM1-14 (4mg/kg) n = 6 Average Tumor Size (mm3) ± SEM  on Day 21 CD3-bindingcontrol H4SH17664D (0.24 1834.87 ± 639.56 mg/kg) + Isotype (4 mg/kg) n =2 CD3-binding control H4SH17664D (0.24 208.29 ± 91.80 mg/kg) +PD-1-blocking RPM1-14 (4 mg/kg) n = 7 BCMA × CD3 REGN5458 (0.04 mg/kg) +2133.12 ± 129.26 Isotype (4 mg/kg) n = 6 BCMA × CD3 REGN5458 (0.04mg/kg) +  21.13 ± 21.13 PD-1-blocking RPM1-14 (4 mg/kg) n = 7 BCMA × CD3REGN5458 (0.24 mg/kg) + 1225.47 ± 289.39 Isotype (4 mg/kg) n = 6 BCMA ×CD3 REGN5458 (0.24 mg/kg) + 113.69 ± 85.39 PD-1-blocking RPM1-14 (4mg/kg) n = 6 Average Tumor Size (mm3) ± SEM  on Day 24 CD3-bindingcontrol H4SH17664D (0.24 2358.81 ± 0.00  mg/kg) + Isotype (4 mg/kg) n =1 CD3-binding control H4SH17664D (0.24  534.03 ± 205.49 mg/kg) +PD-1-blocking RPM1-14 (4 mg/kg) n = 7 BCMA × CD3 REGN5458 (0.04 mg/kg) +3648.37 ± 536.71 Isotype (4 mg/kg) n = 3 BCMA × CD3 REGN5458 (0.04mg/kg) +  53.52 ± 53.52 PD-1-blocking RPM1-14 (4 mg/kg) n = 7 BCMA × CD3REGN5458 (0.24 mg/kg) + 1493.26 ± 973.01 Isotype (4 mg/kg) n = 2 BCMA ×CD3 REGN5458 (0.24 mg/kg) +  54.29 ± 54.29 PD-1-blocking RPM1-14 (4mg/kg) n = 5 Average Tumor Size (mm3) ± SEM  on Day 28 CD3-bindingcontrol H4SH17664D (0.24 All Animals Euthanized mg/kg) + Isotype (4mg/kg) n = 0 CD3-binding control H4SH17664D (0.24 1196.57 ± 467.34mg/kg) + PD-1-blocking RPM1-14 (4 mg/kg) n = 7 BCMA × CD3 REGN5458 (0.04mg/kg) + All Animals Euthanized Isotype (4 mg/kg) n = 0 BCMA × CD3REGN5458 (0.04 mg/kg) +  141.68 ± 141.68 PD-1-blocking RPM1-14 (4 mg/kg)n = 7 BCMA × CD3 REGN5458 (0.24 mg/kg) + 1371.17 ± 0.00  Isotype (4mg/kg) n = 1 BCMA × CD3 REGN5458 (0.24 mg/kg) +  104.44 ± 104.44PD-1-blocking RPM1-14 (4 mg/kg) n = 5

TABLE 33 Tumor-Free Mice at End of Experiment Number of Mice Tumor- Freeat End of Antibody Treatment Experiment (Day 28) CD3-binding controlH4SH17664D (0.24 0 of 7 mg/kg) + Isotype (4 mg/kg) CD3-binding controlH4SH17664D (0.24 2 of 7 mg/kg) + PD-1-blocking RPM1-14 (4 mg/kg) BCMA ×CD3 REGN5458 (0.04 mg/kg) + 0 of 7 Isotype (4 mg/kg) BCMA × CD3 REGN5458(0.04 mg/kg) + 6 of 7 PD-1-blocking RPM1-14 (4 mg/kg) BCMA × CD3REGN5458 (0.24 mg/kg) + 0 of 6 Isotype (4 mg/kg) BCMA × CD3 REGN5458(0.24 mg/kg) + 4 of 6 PD-1-blocking RPM1-14 (4 mg/kg)

Example 21: Anti-BCMA x Anti-CD3 Bispecific Antibodies ActSynergistically with Anti-PD-1 Antibodies to Enhance Anti-Tumor EfficacyIn Vivo

Similar results were obtained in a second experiment, identical to thatdiscussed above in Example 20, except that the number of mice pergroup=10, and the higher dose of BCMAxCD3 REGN5458 was 0.4 mg/kg. Thespecific treatment groups for the second experiment are shown in Table34, below.

TABLE 34 Treatment Groups Group Bispecific Treatment Antibody n 1H4SH17664D (0.4 mg/kg) Isotype (4 mg/kg) 10 2 H4SH17664D (0.4 mg/kg)RPM1-14 (4 mg/kg) 10 3 REGN5458 (0.04 mg/kg) Isotype (4 mg/kg) 10 4REGN5458 (0.04 mg/kg) RPM1-14 (4 mg/kg) 10 5 REGN5458 (0.4 mg/kg)Isotype (4 mg/kg) 10 6 REGN5458 (0.4 mg/kg) RPM1-14 (4 mg/kg) 10

The results demonstrate that combining REGN5458 plus PD-1 blockadeprovides superior anti-tumor efficacy than either REGN5458 or PD-1blockade alone. In particular, the results demonstrate that at day 21(the last day for which data was collected for all treatment groups) thecombination of BCMAxCD3 bispecific antibody and anti-PD-1 antibodyproduced a synergistic therapeutic effect in the inhibition of tumorgrowth (Table 35, BCMAxCD3 at 0.04 mg/kg and anti-PD-1 at 4 mg/kg).Using a 2-way ANOVA test at day 21, p<0.0001 between (i) REGN5458 (0.04mg/kg)+Isotype and the combination of REGN5458 (0.04 mg/kg)+anti-PD-1antibody (Group 3 vs. Group 4), (ii) anti-PD-1 and the combination ofREGN5458 (0.04 mg/kg)+anti-PD-1 antibody (Group 2 vs. Group 4), (iii)anti-PD-1 and the combination of REGN5458 (0.4 mg/kg)+anti-PD-1 antibody(Group 2 vs. Group 6). As discussed above in Example 20, increasing thedose of BCMAxCD3 bispecific antibody (0.4 mg/kg) in combination withPD-1 blockade resulted in tumor inhibition comparable to the lowerbispecific antibody dose combined with PD-1 blockade in this experiment.The demonstrated synergy with the lower dose bispecific antibody isadvantageous because the use of a lower dose reduces the risk of anyadverse side effects. Similarly, the combination of BCMAxCD3 bispecificantibody and anti-PD-1 antibody showed a synergistic therapeutic effectat both doses of bispecific antibody (0.04 mg/kg and 0.4 mg/kg) in thenumber of tumor-free mice at the end of the experiment (day 25), asshown in Table 36.

TABLE 35 Average Tumor Size at Various Time Points Antibody TreatmentAverage Tumor Size (mm3) ± SEM  on Day 3 CD3-binding control H4SH17664D(0.4 mg/kg) +  9.85 ± 0.61 Isotype (4 mg/kg) n = 10 CD3-binding controlH4SH17664D (0.4 mg/kg) + 13.44 ± 1.44 PD-1-blocking RPM1-14 (4 mg/kg) n= 10 BCMA × CD3 REGN5458 (0.04 mg/kg) + 12.41 ± 2.56 Isotype (4 mg/kg) n= 10 BCMA × CD3 REGN5458 (0.04 mg/kg) +  9.73 ± 1.25 PD-1-blockingRPM1-14 (4 mg/kg) n = 10 BCMA × CD3 REGN5458 (0.4 mg/kg) + 11.22 ± 0.68Isotype (4 mg/kg) n = 10 BCMA × CD3 REGN5458 (0.4 mg/kg) +  9.59 ± 1.78PD-1-blocking RPM1-14 (4 mg/kg) n = 10 Average Tumor Size (mm3) ± SEM on Day 6 CD3-binding control H4SH17664D (0.4 mg/kg) + 40.43 ± 4.07Isotype (4 mg/kg) n = 10 CD3-binding control H4SH17664D (0.4 mg/kg) +44.52 ± 2.80 PD-1-blocking RPM1-14 (4 mg/kg) n = 10 BCMA × CD3 REGN5458(0.04 mg/kg) + 38.79 ± 3.52 Isotype (4 mg/kg) n = 10 BCMA × CD3 REGN5458(0.04 mg/kg) + 36.42 ± 3.51 PD-1-blocking RPM1-14 (4 mg/kg) n = 10 BCMA× CD3 REGN5458 (0.4 mg/kg) + 16.11 ± 1.27 Isotype (4 mg/kg) n = 10 BCMA× CD3 REGN5458 (0.4 mg/kg) + 24.34 ± 1.86 PD-1-blocking RPM1-14 (4mg/kg) n = 10 Average Tumor Size (mm3) ± SEM  on Day 10 CD3-bindingcontrol H4SH17664D (0.4 mg/kg) + 149.41 ± 17.08 Isotype (4 mg/kg) n = 10CD3-binding control H4SH17664D (0.4 mg/kg) + 107.34 ± 13.73PD-1-blocking RPM1-14 (4 mg/kg) n = 10 BCMA × CD3 REGN5458 (0.04mg/kg) + 116.32 ±19.99  Isotype (4 mg/kg) n = 10 BCMA × CD3 REGN5458(0.04 mg/kg) + 23.48 ± 3.24 PD-1-blocking RPM1-14 (4 mg/kg) n = 10 BCMA× CD3 REGN5458 (0.4 mg/kg) + 24.27 ± 6.74 Isotype (4 mg/kg) n = 10 BCMA× CD3 REGN5458 (0.4 mg/kg) +  3.60 ± 1.92 PD-1-blocking RPM1-14 (4mg/kg) n = 10 Average Tumor Size (mm3) ± SEM  on Day 13 CD3-bindingcontrol H4SH17664D (0.4 mg/kg) + 386.55 ± 48.49 Isotype (4 mg/kg) n = 10CD3-binding control H4SH17664D (0.4 mg/kg) + 186.87 ± 41.06PD-1-blocking RPM1-14 (4 mg/kg) n = 10 BCMA × CD3 REGN5458 (0.04mg/kg) + 319.91 ± 53.05 Isotype (4 mg/kg) n = 10 BCMA × CD3 REGN5458(0.04 mg/kg) + 10.60 ± 2.34 PD-1-blocking RPM1-14 (4 mg/kg) n = 10 BCMA× CD3 REGN5458 (0.4 mg/kg) +  50.93 ± 20.00 Isotype (4 mg/kg) n = 10BCMA × CD3 REGN5458 (0.4 mg/kg) +  0.74 ± 0.74 PD-1-blocking RPM1-14 (4mg/kg) n = 10 Average Tumor Size (mm3) ± SEM  on Day 18 CD3-bindingcontrol H4SH17664D (0.4 mg/kg) + 1809.29 ± 242.64 Isotype (4 mg/kg) n =9  CD3-binding control H4SH17664D (0.4 mg/kg) +  688.52 ± 152.20PD-1-blocking RPM1-14 (4 mg/kg) n = 10 BCMA × CD3 REGN5458 (0.04mg/kg) + 1314.27 ± 211.22 Isotype (4 mg/kg) n = 10 BCMA × CD3 REGN5458(0.04 mg/kg) +  6.28 ± 4.55 PD-1-blocking RPM1-14 (4 mg/kg) n = 10 BCMA× CD3 REGN5458 (0.4 mg/kg) +  248.51 ± 107.21 Isotype (4 mg/kg) n = 10BCMA × CD3 REGN5458 (0.4 mg/kg) +  3.93 ± 2.67 PD-1-blocking RPM1-14 (4mg/kg) n = 10 Average Tumor Size (mm3) ± SEM  on Day 21 CD3-bindingcontrol H4SH17664D (0.4 mg/kg) + 3094.87 ± 482.38 Isotype (4 mg/kg) n =8  CD3-binding control H4SH17664D (0.4 mg/kg) + 1425.22 ± 338.49PD-1-blocking RPM1-14 (4 mg/kg) n = 10 BCMA × CD3 REGN5458 (0.04mg/kg) + 2446.35 ± 395.48 Isotype (4 mg/kg) n = 10 BCMA × CD3 REGN5458(0.04 mg/kg) +  15.03 ± 10.35 PD-1-blocking RPM1-14 (4 mg/kg) n = 10BCMA × CD3 REGN5458 (0.4 mg/kg) +  453.43 ± 174.75 Isotype (4 mg/kg) n =10 BCMA × CD3 REGN5458 (0.4 mg/kg) +  9.34 ± 7.59 PD-1-blocking RPM1-14(4 mg/kg) n = 10 Average Tumor Size (mm3) ± SEM  on Day 25 CD3-bindingcontrol H4SH17664D (0.4 mg/kg) + Animals Euthanized Isotype (4 mg/kg) n= 0  CD3-binding control H4SH17664D (0.4 mg/kg) + 1918.27 ± 571.19PD-1-blocking RPM1-14 (4 mg/kg) n = 6  BCMA × CD3 REGN5458 (0.04mg/kg) + 2411.64 ± 451.96 Isotype (4 mg/kg) n = 3  BCMA × CD3 REGN5458(0.04 mg/kg) +  38.96 ± 21.47 PD-1-blocking RPM1-14 (4 mg/kg) n = 10BCMA × CD3 REGN5458 (0.4 mg/kg) +  661.70 ± 331.60 Isotype (4 mg/kg) n =8  BCMA × CD3 REGN5458 (0.4 mg/kg) +  32.02 ± 24.67 PD-1-blockingRPM1-14 (4 mg/kg) n = 10

TABLE 36 Tumor-Free Mice at End of Experiment Number of Mice Tumor-Antibody Free at End of Treatment Experiment (Day 25) CD3-bindingcontrol H4SH17664D (0.4 0 of 10 mg/kg) + Isotype (4 mg/kg) CD3-bindingcontrol H4SH17664D (0.4 1 of 10 mg/kg) + PD-1-blocking RPM1-14 (4 mg/kg)BCMA × CD3 REGN5458 (0.04 mg/kg) + 0 of 10 Isotype (4 mg/kg) BCMA × CD3REGN5458 (0.04 mg/kg) + 7 of 10 PD-1-blocking RPM1-14 (4 mg/kg) BCMA ×CD3 REGN5458 (0.4 mg/kg) + 2 of 10 Isotype (4 mg/kg) BCMA × CD3 REGN5458(0.4 mg/kg) + 8 of 10 PD-1-blocking RPM1-14 (4 mg/kg)

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

1. An isolated bispecific antigen binding molecule comprising: (a) afirst antigen-binding domain that specifically binds a human B cellmaturation antigen (BCMA) on a target tumor cell, with an EC₅₀ of lessthan about 100 nM as measured by an in vitro FACS binding assay; and (b)a second antigen-binding domain that specifically binds human CD3 withan EC₅₀ of less than about 10⁻⁶ M as measured by an in vitro FACSbinding assay.
 2. The isolated bispecific antigen binding molecule ofclaim 1, wherein: (a) the bispecific antigen binding molecule activatesT cells in vitro with an EC₅₀ of less than about 10⁻⁹ M; (b) thebispecific antigen-binding molecule mediates in vitro T cell killing oftumor cell lines expressing BCMA with an EC₅₀ of less than about 10⁻⁹ M;(c) the bispecific antigen-binding molecule mediates in vitro autologousT cell killing of primary myeloma cells expressing BCMA with an EC₅₀ ofless than about 10⁻⁸ M; (d) the bispecific antigen-binding moleculeinteracts with amino acid residues 1 through 43 of BCMA as set forth inSEQ ID NO:106; (e) the target tumor cell is a plasma cell; (f) thetarget tumor cell is from a patient suffering from multile mveloma, orfrom another B-cell disorder characterized in part as having B cellsexpressing BCMA; (g) the bispecific antigen-binding moleculecross-reacts with cynomolgus BCMA; (h) the bispecific antigen-bindingmolecule does not cross-react with cynomolgus BCMA; (i) the bispecificantigen-binding molecule inhibits the proliferation of BCMA expressingtumor cells at a dose of from about 0.04 mg/kg to about 4.0 mg/kg; (j)the bispecific antigen-binding molecule inhibits the proliferation ofBCMA+ tumor cells selected from the group consisting of myeloma cells,lymphoma cells and leukemia cells: or (k) the bispecific antigen-bindingmolecule inhibits the proliferation of BCMA+ tumor cells selected fromthe group consisting of H929 cells, MOLP-8 cells and OPM cells. 3-12.(canceled)
 13. The isolated bispecific antigen binding molecule of claim1, wherein the first antigen-binding domain comprises: (a) three heavychain complementarity determining regions (HCDR1, HCDR2 and HCDR3)contained within a heavy chain variable region (HCVR) comprising theamino acid sequence of SEQ ID NO: 66; and (b) three light chaincomplementarity determining regions (LCDR1, LCDR2 and LCDR3) containedwithin a light chain variable region (LCVR) comprising the amino acidsequence of SEQ ID NO:82.
 14. The isolated bispecific antigen bindingmolecule of claim 13, comprising a HCDR1 comprising the amino acidsequence of SEQ ID NO:68, a HCDR2 comprising the amino acid sequence ofSEQ ID NO:70, and a HCDR3 comprising the amino acid sequence of SEQ IDNO:72.
 15. The isolated bispecific antigen-binding molecule of claim 13,comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO:84, aLCDR2 comprising the amino acid sequence of SEQ ID NO:86, and a LCDR3comprising the amino acid sequence of SEQ ID NO:88.
 16. The isolatedbispecific antigen-binding molecule of claim 13, wherein the firstantigen-binding domain comprises a HCVR comprising the amino acidsequence of SEQ ID NO: 66, and a LCVR comprising the amino acid sequenceof SEQ ID NO:
 82. 17. The isolated bispecific antigen-binding moleculeof claim 1, wherein the second antigen-binding domain comprises: (a)three heavy chain complementarity determining regions (HCDR1, HCDR2 andHCDR3) contained within a heavy chain variable region (HCVR) comprisingthe amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 98; and (b) threelight chain complementarity determining regions (LCDR1, LCDR2 and LCDR3)contained within a light chain variable region (LCVR) comprising theamino acid sequence of SEQ ID NO:82.
 18. The isolated bispecific antigenbinding molecule of claim 17, wherein the second antigen-binding domaincomprises: (a) a HCDR1 comprising the amino acid sequence of SEQ ID NO:92 or SEQ ID NO: 100; (b) a HCDR2 comprising the amino acid sequence ofSEQ ID NO: 94 or SEQ ID NO: 102; and (c) a HCDR3 comprising the aminoacid sequence of SEQ ID NO: 96 or SEQ ID NO:
 104. 19. The isolatedbispecific antigen-binding molecule of claim 17, wherein the secondantigen-binding domain comprises a LCDR1 comprising the amino acidsequence of SEQ ID NO:84, a LCDR2 comprising the amino acid sequence ofSEQ ID NO:86, and a LCDR3 comprising the amino acid sequence of SEQ IDNO:88.
 20. The isolated bispecific antigen binding molecule of claim 19,wherein the second antigen-binding domain comprises: (a) HCDR1, HCDR2,HCDR3 domains, respectively, comprising the amino acid sequences of SEQID NOs: 92, 94, 96; and LCDR1, LCDR2, LCDR3 domains, respectively,comprising the amino acid sequences of SEQ ID NOs: 84, 86, 88; or (b)HCDR1, HCDR2, HCDR3 domains, respectively, comprising the amino acidsequences of SEQ ID NOs: 100, 102, 104; and LCDR1, LCDR2, LCDR3 domains,respectively, comprising the amino acid sequences of SEQ ID NOs: 84, 86,88.
 21. The isolated bispecific antigen binding molecule of claim 20,wherein the second antigen-binding domain comprises: (a) a HCVRcomprising the amino acid sequence of SEQ ID NO: 90, and a LCVRcomprising the amino acid sequence of SEQ ID NO: 82; or (b) a HCVRcomprising the amino acid sequence of SEQ ID NO: 98, and a LCVRcomprising the amino acid sequence of SEQ ID NO:
 82. 22. An isolatedbispecific antigen-binding molecule, comprising: (a) a firstantigen-binding domain that comprises HCDR1, HCDR2, HCDR3 domains,respectively, comprising the amino acid sequences of SEQ ID NOs: 68, 70,72, and LCDR1, LCDR2, LCDR3 domains, respectively, comprising the aminoacid sequences of SEQ ID NOs: 84, 86, 88; and (b) a second antigenbinding domain that comprises HCDR1, HCDR2, HCDR3 domains, respectively,comprising the amino acid sequences of SEQ ID NOs: 92, 94, 96, andLCDR1, LCDR2, LCDR3 domains, respectively, comprising the amino acidsequences of SEQ ID NOs: 84, 86,
 88. 23. An isolated bispecificantigen-binding molecule, comprising: (a) a first antigen-binding domainthat comprises HCDR1, HCDR2, HCDR3 domains, respectively, comprising theamino acid sequences of SEQ ID NOs: 68, 70, 72, and LCDR1, LCDR2, LCDR3domains, respectively, comprising the amino acid sequences of SEQ IDNOs: 84, 86, 88; and (b) a second antigen binding domain that comprisesHCDR1, HCDR2, HCDR3 domains, respectively, comprising the amino acidsequences of SEQ ID NOs: 100, 102, 104, and LCDR1, LCDR2, LCDR3 domains,respectively, comprising the amino acid sequences of SEQ ID NOs: 84, 86,88.
 24. The isolated bispecific antigen-binding molecule of claim 22,comprising: (a) a first antigen binding domain that comprises a HCVRcomprising the amino acid sequence of SEQ ID NO: 66, and a LCVRcomprising the amino acid sequence of SEQ ID NO: 82; and (b) a secondantigen binding domain that comprises a HCVR comprising the amino acidsequence of SEQ ID NO: 90, and a LCVR comprising the amino acid sequenceof SEQ ID NO:
 82. 25. The isolated bispecific antigen-binding moleculeof claim 23, comprising: (a) a first antigen binding domain thatcomprises a HCVR comprising the amino acid sequence of SEQ ID NO: 66,and a LCVR comprising the amino acid sequence of SEQ ID NO: 82; and (b)a second antigen binding domain that comprises a HCVR comprising theamino acid sequence of SEQ ID NO: 98, and a LCVR comprising the aminoacid sequence of SEQ ID NO:
 82. 26. An isolated bispecificantigen-binding molecule, comprising: (a) a first antigen-binding domainthat specifically binds human BCMA, and comprises the CDRs of a HCVRcomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 2, 18, 34, 50, 66, 122, and 124, and the CDRs of a LCVRcomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 10, 26, 42, 58, 74, 82, 123, and 125; and (b) a secondantigen-binding domain that specifically binds human CD3.
 27. Theisolated bispecific antigen-binding molecule of claim 26, wherein thefirst antigen-binding domain comprises the CDRs from a HCVR/LCVR aminoacid sequence pair selected from the group consisting of SEQ ID NOs:2/10, 18/26, 34/42, 50/58, 66/74, 122/123, 124/125, 2/82, 18/82, 34/82,50/82, 66/82, 122/82, and 124/82.
 28. The isolated bispecificantigen-binding molecule of claim 27, wherein the first antigen-bindingdomain comprises HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains,respectively, selected from the group consisting of SEQ ID NOs:4-6-8-12-14-16, 20-22-24-28-30-32, 36-38-40-44-46-48, 52-54-56-60-62-64,68-70-72-76- 78-80, 4-6-8-84-86-88, 20-22-24-84-86-88,36-38-40-84-86-88, 52-54-56-84-86-88, and 68-70-72-84-86-88.
 29. Theisolated bispecific antigen-binding molecule of claim 28, wherein thefirst antigen-binding domain comprises the a HCVR/LCVR amino acidsequence pair selected from the group consisting of SEQ ID NOs: 2/10,18/26, 34/42, 50/58, 66/74, 122/123, 124/125, 2/82, 18/82, 34/82, 50/82,66/82, 122/82, and 124/82.
 30. The isolated bispecific antigen-bindingmolecule of claim 26, wherein the second antigen-binding domaincomprises the CDRs of a HCVR/LCVR amino acid sequence pair selected fromthe group consisting of SEQ ID NOs: 90/82 and 98/82.
 31. The isolatedbispecific antigen binding molecule of claim 1 that: (a) competes forbinding to BCMA, or binds to the same epitope on BCMA as a referenceantibody, wherein the reference antibody comprises a firstantigen-binding domain comprising an HCVR/LCVR pair comprising the aminoacid sequences of SEQ ID NOs: 66/82 and a second antigen-binding domaincomprising an HCVR/LCVR pair comprising the amino acid sequences ofeither SEQ ID NOs: 90/82 or SEQ ID NOs: 98/82; or (b) competes forbinding to human CD3, or binds to the same epitope on human CD3 as areference antibody, wherein the reference antibody comprises a firstantigen-binding domain comprising an HCVR/LCVR pair comprising the aminoacid sequences of SEQ ID NOs: 66/82 and a second antigen-binding domaincomprising an HCVR/LCVR pair comprising the amino acid sequences ofeither SEQ ID NOs: 90/82 or SEQ ID NOs: 98/82.
 32. (canceled)
 33. Theisolated bispecific antigen-binding molecule of claim 1 that is abispecific antibody.
 34. The isolated bispecific antigen-bindingmolecule of claim 33, wherein the bispecific antibody comprises a humanIgG heavy chain constant region, optionally wherein (a) the human IgGheavy chain constant region is isotype IgG1 or isotype IgG4, or (b) thebispecific antibody comprises a chimeric hinge that reduces Fcγ receptorbinding relative to a wild-type hinge of the same isotype. 35-37.(canceled)
 38. A pharmaceutical composition comprising the bispecificantigen-binding molecule of claim 1, and a pharmaceutically acceptablecarrier or diluent.
 39. A nucleic acid molecule comprising a nucleotidesequence encoding a bispecific antigen-binding molecule of claim
 1. 40.An expression vector comprising the nucleic acid molecule of claim 39.41. A host cell comprising the expression vector of claim
 40. 42. Amethod of inhibiting growth of a plasma cell tumor in a subject,comprising administering an isolated bispecific antigen-binding moleculeof claim 1 to the subject. 43-45. (canceled)
 46. A method of treating apatient suffering from multiple myeloma, or from another BCMA-expressingB cell malignancy comprising administering an isolated bispecificantigen-binding molecule of claim 1 to the subject. 47-49. (canceled)50. A method of treating a patient suffering from a BCMA-expressingtumor, comprising administering an isolated bispecific antigen-bindingmolecule of claim 1 to the subject in combination with an anti-PD-1antibody or antigen-binding fragment thereof. 51-56. (canceled)